Micropropagation, Somatic Embryogenesis, and Haploid Induction in Passiflora: Advances, Biological Constraints, and Breeding Prospects

Abstract

The genus Passiflora includes species important for fruit production, ornamental value, and breeding programs. Conventional methods, such as seed propagation and vegetative cuttings, face challenges like genetic heterogeneity, pathogen transmission, and long juvenile phases, limiting large-scale cultivation and breeding efficiency. In vitro culture technologies are essential for clonal propagation, germplasm conservation, and improving Passiflora species using biotechnology. This review critically evaluates current progress in micropropagation and regeneration systems in Passiflora spp. and examines the prospects of haploid and doubled haploid technologies as future breeding tools. Unlike previous reviews, which primarily focus on summarizing tissue culture protocols, this study integrates regeneration biology, developmental constraints, and emerging biotechnological approaches to provide a broader framework for research. Additionally, this review offers a comparative analysis of various regeneration systems across Passiflora species and highlights the challenges of genotype-dependent methods. By synthesizing recent advancements in haploid technology, it provides new insights into the potential for accelerating breeding programs in Passiflora, a field where robust protocols are still lacking.

1. Introduction

Passiflora includes over 500 species grown globally for their fruit, ornamental value, and medicinal properties, native to Central and South America. Among these species, P. edulis is of paramount economic importance due to its high commercial value and extensive use in fresh fruit consumption and juice production [1,2,3,4].

Commercial passion fruit production depends heavily on uniform and healthy planting material; however, conventional propagation methods often fail to ensure genetic stability and phenotypic consistency [5,6,7]. Seed propagation results in variability in vine vigor, flowering behavior, and fruit quality due to the high heterozygosity and cross-pollinated reproductive biology of Passiflora species [1,3,8]. Vegetative propagation through stem cuttings or grafting can partially maintain genetic fidelity; however, these approaches often exhibit low rooting efficiency, seasonal limitations, and the risk of systemic pathogen transmission [4,9].

Micropropagation and related tissue culture techniques have been widely applied in Passiflora species, producing healthy and disease-free plantlets in several studies [4,10]. However, reported responses to in vitro culture conditions vary between species and genotypes, and many authors emphasize the need for optimized, species-specific protocols to achieve consistent regeneration outcomes [1,11]. These protocols show varying success across different species and genotypes, indicating the need for species-specific optimization for broader application in commercial propagation and breeding [1,5,12,13]. Somatic embryogenesis in P. edulis shows potential for clonal propagation but faces challenges in embryo-to-plantlet conversion, with variability across genotypes, emphasizing the need for optimized, genotype-specific approaches to improve overall regeneration efficiency [1,14]. Nodal segment-based systems, such as those for P. edulis f. flavicarpa, have shown reliable shoot proliferation on MS medium with BAP, making axillary bud-based micropropagation the most reproducible and transferable method [5,6,13,15].

However, regeneration responses across the genus frequently show strong genotype dependency, reflecting differences in endogenous hormone balance, phenolic metabolism, and stress-response capacity rather than culture medium composition alone [13,15,16]. Alternative regeneration pathways, including de novo organogenesis and somatic embryogenesis, may provide advantages for large-scale propagation, genetic transformation, and synthetic seed production. Somatic embryogenesis is particularly attractive due to its potential scalability and compatibility with bioreactor systems [17,18,19]. Nevertheless, in Passiflora, these systems remain less reproducible than nodal micropropagation and often exhibit low induction frequencies, asynchronous embryo development, and inconsistent embryo-to-plantlet conversion [13,17,20,21].

Oxidative browning due to phenolic compounds released during explant wounding remains a persistent challenge. Although antioxidants such as ascorbic acid and adsorbents including polyvinylpyrrolidone (PVP) or activated charcoal are commonly used to mitigate phenolic oxidation [5,13,17,22], their effectiveness varies among genotypes and may also affect nutrient and growth-regulator availability in culture media. Consequently, improving regeneration efficiency requires a deeper understanding of the physiological and biochemical mechanisms regulating tissue culture responses in this genus.

In parallel with clonal propagation, haploid and doubled haploid (DH) technologies have transformed breeding programs in many crops by enabling the rapid production of completely homozygous lines through androgenesis or isolated microspore culture [23,24,25,26]. No stable haploid induction system has been established in Passiflora, despite its long juvenile phases and high heterozygosity, which make DH technologies valuable for breeding [1,27,28].

Although many studies report regeneration events, most remain descriptive and lack systematic evaluation of reproducibility across genotypes. Consequently, it remains difficult to distinguish regeneration systems that are commercially transferable from those that remain experimental. Therefore, the objective of this review is to critically synthesize current advances in Passiflora micropropagation, somatic embryogenesis, and haploid induction research, with particular emphasis on biological constraints, reproducibility, and translational potential for breeding applications. By integrating physiological, developmental, and biotechnological perspectives, this review aims to provide a framework for improving regeneration efficiency and supporting sustainable genetic improvement of passion fruit and ornamental Passiflora species.

To provide a structured and analytical perspective, this review addresses the following key questions:

(i)

Which regeneration systems in Passiflora are most reproducible across genotypes?

(ii)

What are the main biological constraints limiting regeneration efficiency?

(iii)

Why has haploid induction not yet been successfully established in Passiflora?

(iv)

How can current experimental approaches be translated into practical breeding applications?

Based on this synthesis, this review proposes that regeneration efficiency in Passiflora is governed by the interaction between genotype-specific developmental competence, physiological state of explants, and stress-response regulation, rather than by culture media composition alone. This conceptual perspective provides a mechanistic framework for distinguishing experimentally successful protocols from those with true translational and commercial potential.

2. Review Methodology

2.1. Literature Search Strategy

This review synthesizes critical narrative research on in vitro micropropagation, regeneration systems, and haploid induction in Passiflora species, with a particular focus on identifying the most successful protocols, the role of genotype, and the limitations that influence regeneration efficiency. The goal is to identify gaps in research, assess protocol effectiveness, and outline future directions for Passiflora breeding. Relevant literature was retrieved from major scientific databases, including Web of Science, Scopus, PubMed, and Google Scholar. Keywords used in the search included combinations of Passiflora, passion fruit, micropropagation, in vitro regeneration, somatic embryogenesis, organogenesis, anther culture, microspore culture, androgenesis, gynogenesis, and doubled haploid.

2.2. Inclusion and Exclusion Criteria

Only peer-reviewed articles published in English, reporting on successful protocols and regeneration efficiency, were included. Studies with detailed methodologies, regeneration efficiency data, and genotype effects were prioritized. Publications lacking sufficient methodological clarity or reproducibility were considered only for background context. This ensured a focus on research that has clear implications for practical applications in Passiflora propagation and breeding.

2.3. Data Analysis and Synthesis Approach

The selected studies were analyzed comparatively, focusing on regeneration success rates, genotype-dependent variability, and biological constraints. Data were extracted from studies showing regeneration efficiency, haploid induction success, and PGR treatments across species. Summary tables (

Table 1. Comparative assessment of representative in vitro micropropagation systems in Passiflora spp.

Species/ Genotype	Explant Type	Basal Medium	Key PGR (Plant Growth Regulators) Regime	Reported Regeneration Performance	Reproducibility Across Studies	Cross-Genotype Transferability	Major Technical Constraints	Reference
P. edulis f. flavicarpa	Nodal segments	MS (Murashige and Skoog)	BAP (6-benzylaminopurine) (≈1–2 mg L−1)	Consistent axillary shoot proliferation (≈4–8 shoots per explant per cycle) with reliable rooting	High; reported in several independent studies	Moderate; mainly validated within P. edulis accessions	Cytokinin-induced abnormalities at high concentrations; decline after repeated subcultures	[5,6]
Passiflora foetida	Nodal segments	MS	Cytokinin-based induction (BAP-dominant media)	Shoot regeneration reported but multiplication rates inconsistently quantified	Low–moderate due to limited independent validation	Limited; protocols tested in a few accessions	Strong genotype sensitivity and lack of multi-cycle evaluation	[9]
P. edulis ‘Tainung No. 1’	Nodal segments	MS	Aromatic cytokinins combined with optimized light spectrum	Improved shoot quality and multiplication under controlled light–PGR interactions	Moderate; mainly validated within the tested cultivar	Low; not systematically evaluated across additional genotypes	Requires precise control of light spectrum and hormonal balance	[6,11]
P. edulis (various cultivars)	Nodal segments	MS	BAP-based cytokinin regimes with auxin-supported rooting	Reliable shoot multiplication and rooting reported under optimized culture conditions	Moderate–high across cultivar-level studies	Moderate but genotype-dependent	Phenolic exudation during establishment; contamination from field-derived explants	[8,29]

) and graphs (Figure 1) have been included to illustrate genotype-specific regeneration success and highlight trends in propagation efficiency across studies. Emphasis was placed on identifying consistent trends, major bottlenecks, and key determinants influencing regeneration efficiency and haploid induction potential. Rather than listing protocols, this review synthesizes available evidence to highlight underlying biological mechanisms and practical limitations.

3. Botanical and Horticultural Significance of Passiflora spp.

The genus Passiflora comprises more than 520 recognized species distributed across tropical and subtropical regions and displays remarkable diversity in growth habit, floral morphology, reproductive biology, and ecological adaptation [3,4,30,31]. Economically, the genus is important both as a fruit crop—particularly P. edulis—and as an ornamental plant valued for its distinctive flowers and vigorous climbing habit [1,5,32,33].

In addition to P. edulis, several other species contribute to regional horticulture and niche markets, including P. ligularis (sweet granadilla), P. quadrangularis (giant granadilla), and P. incarnata, which are cultivated for their edible fruits, medicinal properties, or ornamental value [3,4,34,35]. Among these, P. edulis—particularly the purple (P. edulis f. edulis) and yellow (P. edulis f. flavicarpa) forms—remains the most widely cultivated species worldwide due to its fruit quality, adaptability, and high commercial productivity [3,8,27,36].

From a horticultural perspective, successful passion fruit cultivation requires uniform planting material to ensure stable yield, synchronized flowering, and consistent fruit quality. However, seed propagation frequently results in genetic segregation, especially in heterozygous or open-pollinated populations, leading to considerable phenotypic variability among plants [37,38,39]. The high heterozygosity and predominantly cross-pollinated reproductive system of Passiflora further amplify this variability, making clonal propagation essential for maintaining elite cultivars and ensuring production uniformity [3,8,40,41].

Consequently, vegetative propagation methods such as stem cuttings, grafting, and nursery-based propagation systems are widely used to preserve desirable genotypes [3,9]. Nevertheless, these approaches often provide limited multiplication rates and may facilitate the transmission of systemic pathogens when plant material is repeatedly propagated from infected stock plants.

Plant tissue culture technologies have therefore been explored as complementary propagation strategies. Micropropagation enables rapid multiplication, year-round plant production, and the generation of pathogen-free planting material under controlled conditions [1,8,21]. However, the efficiency and scalability of these systems remain variable among species and genotypes, reflecting differences in physiological responses and regeneration competence.

Beyond propagation, the horticultural significance of Passiflora is closely linked to breeding and genetic improvement programs aimed at enhancing yield stability, fruit quality, disease resistance, and tolerance to abiotic stresses such as drought and temperature fluctuations [3,7,27]. In ornamental breeding, selection focuses on floral morphology, pigmentation patterns, and vine architecture, traits that determine the commercial value of ornamental cultivars [42,43,44].

Although haploid and doubled haploid (DH) technologies have not yet been successfully implemented in Passiflora, their application in other horticultural crops demonstrates their potential to accelerate breeding by rapidly generating homozygous lines [23,25,45]. Developing similar technologies in Passiflora could significantly shorten breeding cycles and facilitate the fixation of desirable traits in breeding populations.

Overall, the botanical diversity and horticultural importance of Passiflora highlight the need for efficient propagation systems capable of producing genetically uniform, high-quality planting material on a commercial scale. Improving propagation efficiency while maintaining genetic stability therefore remains a central objective for both passion fruit cultivation and ornamental Passiflora breeding programs.

4. In Vitro Micropropagation of Passiflora spp.

In vitro micropropagation represents one of the most widely used clonal propagation strategies for Passiflora species, enabling rapid multiplication of genetically uniform and pathogen-free planting material under controlled conditions [1,5,6,46]. This approach is particularly valuable for maintaining elite cultivars that cannot be reliably propagated through seed because of genetic segregation and high heterozygosity.

In commercial settings, nodal-based micropropagation in selected P. edulis cultivars can be considered relatively robust, particularly when multiplication and acclimatization protocols are optimized [8,29]. However, extension of these systems to wild species, breeding lines, or ornamental taxa frequently results in reduced multiplication rates and inconsistent rooting responses [1,14,15].

In this review, the reliability of reported micropropagation systems is evaluated based on regeneration efficiency, genotype coverage, reproducibility across independent studies, and successful acclimatization of regenerated plantlets rather than direct experimental validation by the authors. The general workflow of in vitro micropropagation in Passiflora spp.—including explant selection, surface sterilization, culture establishment, shoot multiplication, rooting, and acclimatization—is summarized in Figure 1. The comparative performance of micropropagation systems across different Passiflora species is summarized in Table 1.

4.1. Explant Selection and Preparation

Explant selection plays a critical role in establishing successful in vitro cultures and influencing regeneration efficiency. In Passiflora, nodal segments with pre-existing axillary meristems are the most reliable and widely used explants [5,11,29]. This method minimizes somaclonal variation and preserves genetic integrity, making it the most suitable for large-scale commercial propagation.

Alternative explants—including leaf discs, cotyledonary nodes, internodes, and petioles—have also been explored mainly for organogenesis or callus induction [18,47,48,49]. However, their regeneration responses often depend strongly on genotype and developmental stage, resulting in variable morphogenic outcomes across studies [15,50,51].

Surface sterilization remains a critical challenge during culture initiation. Standard protocols typically involve ethanol followed by sodium hypochlorite treatments [17,52,53]. Nevertheless, endogenous microbial contamination frequently compromises culture establishment when explants are obtained from field-grown plants [5,8,54]. Limited comparative studies quantifying contamination frequencies among explant sources highlight an important methodological gap in Passiflora micropropagation research [15,51,55].

4.2. Culture Media and Plant Growth Regulators

Murashige and Skoog (MS) medium remains the most widely used basal formulation for Passiflora micropropagation [5,6,29]. Standard supplementation with sucrose (20–30 g L⁻¹) and agar provides the basic nutritional support required for explant growth [17,56,57]. However, comparative evaluations of alternative formulations such as Woody Plant Medium remain limited for this genus [58].

Cytokinins, particularly 6-benzylaminopurine (BAP), play a central role in shoot induction and multiplication. Concentrations between 1 and 2 mg L⁻¹ BAP commonly promote multiple shoot formation in P. edulis [5,6,14]. However, despite the widespread use of BAP-based systems, regeneration responses remain highly variable across studies. This variability suggests that cytokinin concentration alone is not the primary determinant of success, and that genotype-specific responses and the physiological status of explants play more critical roles in regulating regeneration efficiency. For example, nodal explants cultured on MS medium containing 1.5–2.0 mg L⁻¹ BAP can produce several shoots per explant within a single culture cycle [10,59,60]. However, excessive cytokinin exposure may induce physiological disorders such as hyperhydricity, chlorosis, or reduced rooting capacity [56,61,62]. Combined cytokinin regimes, such as BAP with kinetin (KIN) or meta-topolins, have also been investigated to improve shoot quality and reduce physiological abnormalities, although these approaches remain poorly validated across multiple genotypes [11,63,64].

Auxins such as indole-3-butyric acid (IBA) are commonly used to stimulate root induction [1,17,65]. Rooting responses are influenced not only by auxin concentration but also by environmental factors including light intensity, carbohydrate availability, and vessel ventilation [66,67,68].

4.3. Shoot Induction and Multiplication

Nodal explants cultured on cytokinin-supplemented MS medium typically show vigorous shoot proliferation, with multiplication rates of approximately 4–8 shoots per explant per culture cycle under optimized conditions [10,14,59]. However, multiplication efficiency is strongly influenced by genotype, physiological age of the explant, and subculture duration. Long-term stability across repeated subculture cycles is rarely evaluated, and extended subculturing may lead to reduced shoot vigor or culture decline [69,70,71].

Despite these reported successes, direct comparisons among studies remain limited, and multiplication rates are often not evaluated under standardized conditions. As a result, it is difficult to determine whether differences in regeneration efficiency reflect true biological variation or inconsistencies in experimental design.

Two-step multiplication strategies—such as initial high-BAP induction followed by stabilization with BAP and kinetin—have improved shoot proliferation in selected genotypes [15,72]. Cultivar-specific optimization has also been reported in P. edulis ‘Tainung No. 1’, where interactions between aromatic cytokinins and light spectrum improved shoot multiplication and plantlet quality [11]. Nevertheless, the transferability of these protocols across different Passiflora species remains insufficiently validated. For example, genotype-specific responses have been observed in P. edulis f. flavicarpa and P. incarnata, where similar culture media produced different morphogenic responses [1,29,73].

4.4. Rooting and Acclimatization

Root induction is typically achieved on half-strength MS medium supplemented with 1–2 mg L⁻¹ IBA [1,29,74,75]. Rooting percentages exceeding 70–80% have frequently been reported under optimized conditions [60,76]. However, many studies emphasize rooting frequency rather than root system architecture or post-transplant performance.

Root characteristics—including root number, length, and branching—play a crucial role in plantlet survival during acclimatization [66,77,78]. During this phase, plantlets gradually adapt to ex vitro conditions as anatomical features developed in vitro—such as poorly functioning stomata and thin cuticles—adjust to natural environments. Survival rates above 80% have been reported in optimized acclimatization systems, although genotype-dependent variability remains common [14,79]. During acclimatization, plantlets are gradually transferred to ex vitro conditions under controlled environments characterized by high relative humidity (80–90%), reduced light intensity, and gradual exposure to ambient conditions [66,77]. The use of substrates such as peat-perlite mixtures, intermittent misting, and progressive reduction in humidity is commonly employed to improve survival rates [66,77]. Successful acclimatization depends on the development of functional stomata, cuticle formation, and robust root systems, which collectively determine plantlet establishment under greenhouse or field conditions [66,78,79].

4.5. Organogenesis and Alternative Explants

Non-meristematic explants such as petioles, internodes, and cotyledonary nodes have been used for indirect organogenesis in several Passiflora species [9,47,50]. These approaches expand regeneration options for applications including genetic transformation, mutation breeding, and regeneration from juvenile tissues [80,81]. However, callus-mediated regeneration often exhibits greater variability and a higher risk of somaclonal variation compared with nodal micropropagation [82,83]. Consequently, organogenesis should be considered a complementary pathway rather than a replacement for nodal culture systems.

Organogenesis plays a vital role in breeding programs by enabling clonal propagation and the development of disease-resistant cultivars. This method significantly reduces time-to-market for new varieties and improves overall breeding efficiency.

4.6. Persistent Constraints During Culture Establishment

Oxidative browning and microbial contamination remain major constraints in Passiflora micropropagation. Phenolic compounds released from wounded explants frequently cause tissue oxidation, reducing culture establishment and regeneration efficiency [9,15,84]. Antioxidants such as ascorbic or citric acid and adsorbents including activated charcoal or polyvinylpyrrolidone (PVP) are commonly used to mitigate these effects [52,85].

Microbial contamination is another persistent issue, particularly when explants are obtained from field-grown plants containing endophytic microorganisms. Improved sterilization procedures and the use of greenhouse-grown donor plants can reduce contamination rates. Nevertheless, variability in oxidative responses and contamination frequencies across studies highlights ongoing reproducibility challenges in Passiflora micropropagation systems [15,51].

Overall, nodal micropropagation represents the most reproducible and practically applicable regeneration system in Passiflora. Its reliability is largely attributed to the use of pre-existing meristems, which reduces variability and maintains genetic stability [1,5]. In contrast, regeneration systems based on non-meristematic tissues remain highly genotype-dependent and often lack reproducibility. This indicates that the key limitation is not protocol availability, but the lack of standardized validation across multiple genotypes.

Importantly, most published studies report results from single genotypes or limited experimental repetitions, making it difficult to assess true reproducibility and the broader applicability of these protocols [8,52].

5. Somatic Embryogenesis and Advanced Regeneration in Passiflora spp.

Somatic embryogenesis (SE) is a cellular reprogramming process in which somatic cells acquire embryogenic competence and develop into bipolar embryo-like structures capable of regenerating complete plants under in vitro conditions. Unlike organogenesis, which relies on pre-existing meristems, SE involves de novo embryo formation that resembles the developmental sequence of zygotic embryogenesis. Owing to its potential scalability and compatibility with bioreactor systems, SE has been widely regarded as a promising platform for large-scale clonal propagation, synthetic seed production, cryopreservation, and genetic transformation in horticultural crops [4,5,6,86].

Despite these advantages, somatic embryogenesis in Passiflora remains less standardized and reproducible than nodal micropropagation systems. While axillary bud proliferation in selected P. edulis cultivars is relatively reliable, SE protocols across the genus are frequently genotype-dependent and often show variable induction rates and inconsistent embryo-to-plantlet conversion [1,15,87]. Consequently, somatic embryogenesis in Passiflora is still considered largely experimental compared with established micropropagation approaches, highlighting the need for improved understanding of the physiological and molecular factors regulating embryogenic competence [20,86,88].

The principal embryogenic pathways—direct embryogenesis from explant tissues and indirect embryogenesis via callus formation—together with their associated developmental stages and technical bottlenecks are summarized in Figure 2.

5.1. Evidence of Somatic Embryogenesis in Passion Fruit

Somatic embryogenesis in Passiflora is highly genotype-dependent. For example, P. edulis shows high efficiency, while P. foetida fails to produce embryos consistently, highlighting the need for optimized protocols in genotype-specific cases [89,90,91]. Direct SE has been observed in P. edulis using explants such as leaf discs and root tissues cultured with optimized auxin–cytokinin media [8,15,92]. Indirect SE, involving the formation of an embryogenic callus, has been reported in tissues such as immature seeds, leaves, and anther-derived explants from species like P. edulis and P. cincinnata [14,93,94]. However, these methods remain highly genotype-dependent, with inconsistent regeneration rates.

Although these studies confirm the biological feasibility of SE in Passiflora, most protocols remain genotype-specific and lack multi-genotype validation, which limits their reproducibility and broader application [1,21].

5.2. Determinants of Embryogenic Competence

Embryogenic competence refers to the ability of somatic cells to reprogram their developmental fate and initiate embryo formation under inductive culture conditions. In Passiflora, this capacity is influenced by genotype, explant developmental stage, hormonal balance, and oxidative status [1,95,96]. Responses vary considerably among species and accessions, indicating that SE induction is governed by genotype-specific differences in cellular plasticity and stress responsiveness rather than by a single universal culture condition. For example, embryogenic responses have been reported in P. edulis and P. cincinnata, although induction frequency and embryo development efficiency differ among genotypes [15,94,97].

Juvenile tissues, such as young leaves or root segments, typically show higher embryogenic responsiveness due to their greater cellular plasticity [17,98,99]. Hormonal regulation is another key factor: auxin-induced dedifferentiation, commonly using 2,4-D, promotes callus formation and acquisition of embryogenic competence, whereas transfer to cytokinin-containing media supports embryo differentiation. Yet, imbalanced auxin–cytokinin ratios frequently result in excessive callus proliferation without synchronized embryo maturation, suggesting that temporal regulation of PGR exposure is under-optimized in many protocols [6,19,20].

Stress signaling and oxidative pathways may also influence embryogenic responses. For example, additives such as nanoparticles or antioxidants have been reported to enhance embryogenic callus formation, although their reproducibility across genotypes remains uncertain [21,48,100].

Prolonged callus maintenance may further reduce embryogenic potential due to cumulative oxidative stress, hormonal imbalance, and epigenetic instability, ultimately leading to reduced regeneration efficiency [20,69].

5.3. Developmental Progression and Histological Validation

Somatic embryos in Passiflora generally progress through globular, heart, torpedo, and cotyledonary stages similar to those observed during zygotic embryogenesis (Figure 2) [17,20,101]. Histological analyses in P. cincinnata have revealed organized cell division within embryogenic callus prior to embryo differentiation [94,102], and comparable observations have been reported in P. edulis [15,103].

Histological validation is essential for distinguishing true somatic embryos from organogenic structures or non-embryogenic callus masses, as misidentification can lead to overestimation of regeneration efficiency [17,102]. Despite successful embryo induction, conversion of mature embryos into autotrophic plantlets remains a major technical bottleneck. Abnormal embryo morphology, incomplete vascular development, and desiccation sensitivity frequently reduce regeneration success [6,20].

In addition, the molecular regulation of SE in Passiflora remains largely unexplored. In model species, transcription factors such as WUSCHEL (WUS), LEAFY COTYLEDON (LEC), and BABY BOOM (BBM) regulate embryogenic competence [16,102,104], but comparable molecular studies in Passiflora are still limited.

5.4. Applications and Realistic Prospects

Somatic embryogenesis has considerable potential for high-density clonal propagation, synthetic seed production, cryopreservation, and regeneration systems compatible with genetic transformation [1,80,105,106]. However, practical implementation of SE in Passiflora remains constrained by genotype-dependent induction, asynchronous embryo maturation, and variable embryo-to-plantlet conversion efficiency [1,21,107].

Despite these limitations, SE provides a valuable experimental platform for investigating developmental plasticity, regenerating plants from non-meristematic tissues, and supporting future transformation or synthetic seed technologies. At present, SE in Passiflora should therefore be considered biologically feasible but technologically immature compared with nodal micropropagation systems. Advancing SE toward a reliable propagation platform will require multi-genotype validation, standardized reporting of regeneration efficiency, and integration of physiological and molecular analyses. Such efforts will be essential for translating experimental SE protocols into practical biotechnology tools for Passiflora breeding and propagation.

These observations indicate that somatic embryogenesis in Passiflora is constrained by intrinsic biological limitations, including incomplete developmental reprogramming, genotype-dependent responsiveness, and inefficient embryo maturation, rather than by insufficient technical optimization alone [17,18,20]. Therefore, improving SE efficiency requires a deeper understanding of cellular totipotency and regulatory mechanisms rather than further empirical adjustment of culture conditions.

6. Haploid Induction and Microspore Culture: Comparative Perspectives

Haploid and doubled haploid (DH) technologies enable rapid fixation of homozygosity within a generation [23,108]. However, comparable systems have not yet been established in Passiflora [1,3,109]. This represents a major limitation for passion fruit breeding, where long juvenile phases and high heterozygosity slow conventional genetic improvement [3,110,111]. Evaluating haploid induction strategies developed in other crops therefore provides a useful framework for identifying realistic opportunities for implementing similar technologies in Passiflora. By bypassing repeated selfing cycles, DH systems dramatically shorten breeding timelines, accelerate quantitative trait locus (QTL) mapping, and enhance hybrid seed development [24,108]. Haploids may be induced through wide hybridization, gynogenesis, or androgenesis, with androgenesis—via anther or isolated microspore culture—being the most widely implemented method in crops [26,112].

Although somatic embryogenesis provides an effective regeneration pathway, it is distinct from microspore embryogenesis used in doubled haploid (DH) production [17,20]. DH systems rely specifically on microspore-derived embryogenesis following androgenesis, whereas somatic embryogenesis originates from somatic tissues [23,25]. Therefore, while both processes involve embryo formation, their biological origins and applications differ substantially.

6.1. Androgenesis: Biological Basis, Technical Constraints, and Lessons from Model Crop Systems

Androgenesis redirects immature microspores from the gametophytic to the sporophytic developmental pathway, producing haploid plants [25,113]. These haploid plants can then undergo chromosome doubling, either spontaneously or through chemical treatments such as colchicine, resulting in homozygous doubled haploid lines. However, the process is highly dependent on the developmental stage of microspores and the genotype of the donor plant, factors that remain underexplored in Passiflora [23,114].

Isolated microspore culture is generally more efficient than whole-anther culture because it minimizes interference from surrounding somatic tissues and reduces the risk of diploid regeneration [112,113]. A key step in androgenesis is the induction of sporophytic reprogramming, which is typically triggered by abiotic stress treatments such as heat shock, cold pretreatment, or osmotic stress [17,115,116].

However, androgenesis efficiency is strongly influenced by factors including microspore developmental stage, donor plant physiology, genotype, and stress calibration [113,117]. Even in species where androgenesis is well established, induction frequencies can vary widely among genotypes, indicating that embryogenic reprogramming capacity is inherently genotype dependent [112,118].

From a breeding perspective, efficient regeneration platforms are essential for successful doubled haploid production because haploid embryos must survive induction, regenerate into plantlets, and undergo chromosome doubling to produce fertile lines. In Passiflora, the lack of reliable regeneration systems for microspore-derived tissues means that haploid induction and plant regeneration remain closely linked challenges rather than independent steps. Consequently, improving regeneration competence is a prerequisite for developing DH breeding technologies in this genus [119,120,121].

The general workflow of androgenesis-based haploid induction—from donor plant selection to microspore isolation, stress pretreatment, embryo development, and chromosome doubling—is illustrated in Figure 3.

In several crops, including barley, wheat, and rice, androgenesis has reached a relatively advanced stage of technical development. Optimized microspore staging at the late uninucleate stage, combined with controlled temperature pretreatments and auxin-rich induction media, enables reproducible haploid regeneration in responsive genotypes [112,122]. Similarly, in solanaceous crops such as Capsicum annuum, androgenesis has become reproducible in selected breeding lines following extensive optimization of microspore developmental staging and stress-induction protocols [23,113].

These examples demonstrate that successful androgenesis systems typically emerge through systematic genotype screening, precise developmental staging, and careful optimization of culture conditions. However, even well-established systems still face technical limitations, including albino plant formation, embryo abortion, and low regeneration frequency [23,116,120]. For Passiflora, these model systems highlight that successful androgenesis requires coordinated developmental timing, stress-induced reprogramming, and effective regeneration competence rather than simply the presence of a culture protocol.

6.2. Mechanisms of Microspore Embryogenesis

Microspore embryogenesis is now recognized as a stress-induced developmental reprogramming process involving chromatin remodeling, epigenetic regulation, hormonal redistribution, and activation of stress-response pathways [17,20]. Following successful induction, microspores typically develop through globular, heart, and cotyledonary stages resembling those of zygotic embryogenesis, although the underlying regulatory networks differ substantially from sexual embryo development [120,123,124].

Recent studies indicate that reactive oxygen species (ROS) signaling, autophagy, and epigenetic modifications play important roles in the transition from the gametophytic to the sporophytic developmental pathway [19,20,125]. These processes are likely responsible for the strong genotype-dependent responses frequently observed in androgenesis systems. However, the molecular mechanisms regulating microspore embryogenesis have not yet been systematically investigated in Passiflora. Consequently, current assumptions regarding haploid induction feasibility in this genus largely rely on knowledge derived from model crop systems.

6.3. Current Status of Haploid Induction in Passiflora

In contrast to cereals and several horticultural crops, reproducible haploid induction systems have not yet been established in Passiflora species [1,126]. Reports of androgenesis or microspore-derived embryogenesis remain extremely limited, and no study has demonstrated stable haploid regeneration across multiple Passiflora genotypes.

Nevertheless, some evidence indicates that haploid production is biologically possible in this genus. A gynogenesis study in P. edulis cultured unfertilized ovules from 11 genotypes and recovered haploid regenerants (2n = 1x = 9), although the response was strongly genotype-dependent and the highest embryo induction rate reached only 7.67% [126,127]. This finding suggests that haploid development is feasible in Passiflora, but currently occurs through ovule-derived gynogenesis rather than through validated microspore-derived androgenesis.

The absence of androgenesis protocols likely reflects several biological constraints. Passiflora species exhibit perennial growth habits, extended juvenile phases, variable pollen viability, and high phenolic content in reproductive tissues, factors that may interfere with microspore viability and embryogenic induction during in vitro culture. In addition, the developmental staging of microspores in Passiflora has not yet been systematically characterized. In other crops, accurate identification of the late uninucleate microspore stage is essential for successful androgenesis induction [112,113,117]. Without similar developmental studies, attempts to establish androgenesis systems in Passiflora remain largely exploratory. A comparative overview of androgenesis systems in major crops and their potential implications for Passiflora biotechnology is presented in

Table 2. Comparative androgenesis systems in major crops and their translational implications for Passiflora spp.

Crop/System	Culture Type	Key Induction Trigger	Typical Outcome	Technical Maturity	Major Bottleneck	Relevance to Passiflora	Reference
Capsicum annuum (pepper)	Anther culture/isolated microspore culture	Cold pretreatment combined with auxin-rich induction media	Haploid and doubled haploid (DH) plant regeneration reported, though strongly genotype-dependent	Moderately established	Low induction frequency and frequent albino plant formation	Provides methodological guidance for floral bud staging, genotype screening, and optimization of stress pretreatments	[25,113]
Solanum melongena (eggplant)	Anther culture and isolated microspore culture	Heat or cold shock applied to immature pollen	Doubled haploid production protocols developed in selected genotypes	Established but genotype-sensitive	Strong dependence on microspore developmental stage and donor plant physiology	Demonstrates stepwise optimization strategies required for androgenesis in recalcitrant species	[25]
Cereals (Hordeum vulgare, Triticum aestivum, Oryza sativa)	Isolated microspore culture	Heat shock, starvation stress, and optimized induction media	High-frequency haploid embryo production in responsive lines with efficient chromosome doubling	Highly established	Albino plant formation and persistent genotype dependency	Provides a mechanistic framework for stress-induced microspore embryogenesis and haploid production systems	[23,112]
Passiflora spp.	No validated androgenesis system	Not standardized	No reproducible microspore-derived haploid or DH regeneration reported	Exploratory stage	Lack of microspore developmental staging, unknown embryogenic responsiveness, and limited experimental studies	Represents a major research gap; future work should prioritize cytological microspore staging, donor plant conditioning, and stress-induction optimization	[1,15]
P. edulis (gynogenesis evidence)	Unfertilized ovule culture	Ovule culture under controlled in vitro conditions	Haploid embryos recovered and cytologically confirmed (2n = 1x = 9), with genotype-dependent response	Experimental	Low embryo induction frequency and strong genotype dependence	Demonstrates that haploid production is biologically possible in Passiflora, suggesting gynogenesis as a complementary pathway to explore	[127]

.

6.4. Strategic Opportunities and Future Directions

Although haploid induction systems have not yet been established in Passiflora, advances in androgenesis research in other crops provide valuable methodological insights. Successful systems typically combine precise microspore developmental staging, controlled abiotic stress pretreatments, optimized induction media, and reliable chromosome-doubling protocols [23,112].

Future research in Passiflora should therefore prioritize cytological characterization of microspore development, evaluation of pollen viability across genotypes, optimization of stress pretreatments for embryogenic induction, and investigation of hormonal and epigenetic regulation during microspore reprogramming. In addition, gynogenesis should be reconsidered as a complementary strategy. Evidence from ovule culture in P. edulis indicates that haploid production is biologically possible, although induction frequencies remain low [127].

Even moderate success in haploid induction could significantly accelerate breeding programs for passion fruit, where long juvenile phases and high heterozygosity currently constrain genetic improvement [108]. Overall, progress in Passiflora haploid biotechnology will depend on systematic studies addressing developmental staging, induction stress optimization, and genotype-responsive regeneration systems.

6.5. Determinants and Constraints Limiting Haploid Induction in Passiflora

Despite these opportunities, several biological and technical constraints continue to limit the development of reproducible haploid induction systems in Passiflora. Regeneration efficiency often varies substantially among species and cultivars, reflecting genotype-dependent developmental competence, donor plant physiology, and culture conditions [1,17,18]. Understanding these determinants is essential not only for micropropagation but also for advanced regeneration systems such as somatic embryogenesis and androgenesis.

6.6. Genotype Dependency

Different Passiflora species and cultivars frequently exhibit distinct morphogenic responses even under identical culture conditions [1,9,90]. Similar genotype effects have been reported in androgenesis systems of many crops, where only a subset of genotypes responds efficiently to induction treatments [23,112]. However, systematic screening of diverse Passiflora germplasm for regeneration competence remains limited.

6.7. Donor Plant Physiology

The physiological condition of donor plants strongly influences explant responsiveness. Tissues obtained from actively growing greenhouse plants generally exhibit higher regeneration capacity than those collected from stressed field-grown plants [9,52,128]. In androgenesis systems of other crops, donor plant management—including temperature regimes and nutrient availability—has also been shown to affect microspore embryogenic competence [112,113].

6.8. Developmental Stage and Explant Source

Explant type and developmental stage are critical determinants of regeneration efficiency. Nodal segments and shoot tips are commonly used for micropropagation because they contain pre-existing meristematic tissues capable of stable shoot development [9,67,129]. In androgenesis systems, precise identification of the late uninucleate microspore stage is essential for embryogenic induction [21,112,113]. However, detailed cytological characterization of microspore development in Passiflora remains largely unavailable.

6.9. Culture Media and Hormonal Regulation

Culture medium composition influences morphogenic responses through its effects on nutrient availability and hormonal balance. Murashige and Skoog (MS) medium remains the most widely used formulation in Passiflora tissue culture [5,130]. In androgenesis systems, factors such as nitrogen form, carbohydrate concentration, and osmotic regulators play key roles in maintaining microspore viability and promoting embryogenic reprogramming [113,120]. In addition, imbalanced cytokinin–auxin ratios may lead to abnormal morphogenesis or excessive callus formation [5,131,132].

6.10. Stress Signaling and Environmental Factors

Abiotic stress treatments are major triggers of microspore embryogenesis in many crops. Heat shock, cold pretreatment, or osmotic stress can redirect microspores from gametophytic to sporophytic development [17,112,115]. Because androgenesis depends on carefully calibrated stress conditions, identifying species- and genotype-specific induction windows will be critical for establishing haploid systems in Passiflora.

6.11. Oxidative Stress and Phenolic Compounds

Phenolic exudation and oxidative browning are persistent obstacles in Passiflora tissue culture due to the high phenolic content of many species [1,9,84]. Although antioxidants and adsorbents can reduce browning, excessive suppression of oxidative processes may interfere with developmental signaling. Controlled reactive oxygen species (ROS) signaling has been shown to promote embryogenic reprogramming in several species [17,20].

6.12. Genetic and Epigenetic Regulation

Regeneration competence is also controlled by genetic and epigenetic mechanisms, including DNA methylation, chromatin remodeling, and transcriptional regulation [20,133]. In model species, transcription factors such as WUSCHEL, BABY BOOM, and LEAFY COTYLEDON regulate embryogenic competence [19,133]. However, comparable molecular studies in Passiflora remain scarce, limiting understanding of the regulatory networks underlying morphogenic responses.

6.13. Synthesis

Overall, the major constraints for haploid induction in Passiflora include genotype dependency, donor plant conditioning, microspore developmental staging, stress calibration, and limited knowledge of molecular regulation. Addressing these factors will be essential for developing reproducible androgenesis-based haploid systems in this genus.

The absence of a successful androgenesis system in Passiflora indicates that haploid induction is constrained by multiple interacting factors, including microspore developmental stage, stress response capacity, and regeneration competence. Unlike other crops, haploid production in Passiflora cannot be treated as an independent process but must be integrated with efficient regeneration systems [23,26]. Therefore, future research should adopt a stepwise approach combining cytological analysis, stress-induced reprogramming, and regeneration optimization.

Furthermore, most current evidence in Passiflora is derived from indirect observations or analogy with model crop systems rather than from direct experimental validation. This highlights a critical knowledge gap and emphasizes the need for species-specific studies focusing on microspore developmental biology and stress-induced reprogramming.

7. Applications and Translational Potential of In Vitro Technologies in Passiflora

In vitro propagation technologies play an important role in the improvement, conservation, and commercialization of Passiflora species [3,8]. Among available approaches, nodal micropropagation is currently the most reliable and reproducible system, particularly for selected cultivars of P. edulis. In contrast, somatic embryogenesis and haploid technologies remain developmental strategies whose practical application depends on improved reproducibility and genotype responsiveness [1,9,10].

7.1. Rapid Clonal Propagation and Clean Stock Production

Nodal micropropagation enables rapid multiplication of genetically uniform planting material and represents the most widely adopted in vitro technique for passion fruit propagation. Repeated subculture cycles can produce large numbers of plants while preserving important horticultural traits such as fruit quality and flowering behavior [4,9]. Micropropagated plants generally perform comparably to conventionally propagated material when acclimatization protocols are optimized [52,134]. Meristem-based culture can also reduce the transmission of systemic pathogens associated with vegetative propagation [1,8]. However, broader multi-genotype validation is still required to confirm the scalability of these systems [135].

7.2. Germplasm Conservation

In vitro culture also provides valuable tools for conserving Passiflora genetic resources. Techniques such as slow-growth storage and embryogenic culture systems can maintain plant material under controlled conditions and reduce the risk of genetic erosion in field collections [1,8]. Embryogenic cultures may additionally support cryopreservation-based germplasm storage [15,136]. Nevertheless, cryopreservation protocols remain poorly standardized across Passiflora species and require further optimization.

7.3. Breeding and Biotechnological Applications

Efficient regeneration systems form the foundation for modern plant breeding technologies. In Passiflora, in vitro regeneration supports mutation breeding, somaclonal variation screening, and genetic transformation [1,3]. Haploid and doubled haploid technologies could further accelerate breeding by enabling the rapid fixation of homozygous lines [23,25]. However, reproducible androgenesis systems have not yet been developed in Passiflora.

Recent advances in genome editing and computational optimization may enhance future applications. Genome editing tools such as CRISPR/Cas systems require efficient regeneration platforms for successful implementation [137,138]. In addition, machine learning–based modeling approaches have been proposed for predicting regeneration responses and optimizing culture conditions in genotype-sensitive systems [47,139].

7.4. Translational Perspective

The immediate applications of in vitro technologies in Passiflora include elite clone multiplication, cleaner nursery stock production, and germplasm conservation. By contrast, somatic embryogenesis, genome editing, and haploid technologies remain promising long-term strategies whose broader impact will depend on improved reproducibility and stronger integration between research laboratories and commercial propagation systems.

From a practical perspective, regeneration systems in Passiflora can be categorized into three levels of technological maturity. Nodal micropropagation is well-established and suitable for commercial use. Somatic embryogenesis remains intermediate, with potential but limited reproducibility. In contrast, haploid and doubled haploid technologies are still at an exploratory stage and require further development before practical application.

8. Challenges and Future Directions in Passiflora Tissue Culture

The future of Passiflora tissue culture will depend heavily on the development of more robust and reproducible protocols. Genotype-specific challenges must be addressed through the integration of modern biotechnological tools, such as genome editing and data-driven culture optimization [16,17]. Additionally, improving the multi-genotype validation of regeneration systems will be key to making these technologies scalable for breeding applications. The integration of genome editing and advanced optimization techniques will accelerate the development of reliable propagation systems that can be applied across diverse species and genotypes [1,5].

At the biological level, further investigation into the mechanisms underlying somatic embryogenesis and organogenesis is required, particularly the roles of hormonal balance, oxidative stress, and developmental regulation [18,20]. Integrating molecular approaches, including transcriptomic and hormonal analyses, can provide valuable insights into the factors controlling regeneration competence [16].

The lack of a reliable androgenesis system is a major limitation in Passiflora breeding. Establishing efficient haploid induction systems is a priority for accelerating genetic improvement [23,26]. Addressing these challenges will require a coordinated approach that combines physiological, molecular, and technical advancements to bridge the gap between experimental research and practical applications.

9. Conclusions

In vitro regeneration technologies are vital tools for the propagation, conservation, and genetic improvement of Passiflora species. Among the available techniques, nodal micropropagation via axillary bud proliferation stands out as the most reliable and widely applicable, particularly for selected P. edulis genotypes. However, this method’s success can vary significantly across different species and genotypes, emphasizing the need for continued optimization.

Somatic embryogenesis and haploid technologies show promise for accelerating breeding programs but remain inconsistent and genotype-dependent, limiting their practical application in Passiflora. Although advances in these areas are ongoing, establishing standardized protocols for these methods should be prioritized.

Breeders are encouraged to integrate somatic embryogenesis and haploid induction technologies into their programs to improve genetic uniformity and reduce breeding timelines. Moreover, to move beyond empirical protocols, future research should focus on a deeper understanding of the physiological and molecular mechanisms behind regeneration, ensuring multi-genotype validation and reproducibility.

Advances in genome editing and data-driven optimization approaches will also be instrumental in enhancing regeneration efficiency and facilitating genetic improvement. Distinguishing reliable propagation systems from experimental methods is key to translating laboratory advances into sustainable passion fruit production and genetic improvement.