Introduction

Piper nigrum L. (Piperaceae) (2n = 4x = 52), commonly known as black pepper, is referred to as the ‘king of spices’ due to its widespread use and the quantity of trade and commerce among spices in the international market (Srinivasan 2007). This robust vine thrives across diverse altitudes and exhibits remarkable adaptability to a variety of climatic gradients (Ravindran and Kallupurackal 2012). The name ‘black pepper’ derives from the distinctive coloration of its economically valued peppercorn seeds. The plant is native to India and traces its origin from the wet humid regions of the WesternGhats in southern India. Globally, the black pepper is cultivated on a total area of 678,215 hectares with the production of 793,817.98 tons (FAO 2021). India with the production of 64,815.81 tons in the cultivated area of 131,711 hectares stands out as the second leading country after Indonesia in area under cultivation and fifth in production, contributing over 8.16% of the world production (FAO 2021), with 17,958 tons earmarked for export, contributing INR 7.26 billion to the nations’ foreign exchange reserves (SBI 2022). In India, Karnataka is the major producer contributing over 60.46% of the production in the country followed by Kerala (APEDA 2021).

The genus Piper comprises 2048 recognized species (POWO 2023), although it is P. nigrum, P. longum L. and P. betle L. are the three most well-known and economically important ones. Within the Indian subcontinent, a rich diversity of 114 Piper species exists, with 18 species nestled within the sub-mountainous Western Ghats, the contiguous peninsula, and the coastal regions (Nirmal Babu et al. 2015). The critical conservation status of P. nigrum becomes evident as it is listed as endangered in Andhra Pradesh (2001), near threatened in Karnataka (1999) and Tamil Nadu (1998), as revealed by the Conservation Assessment and Management Prioritization (CAMP) workshops (Gowthami et al. 2021a).

As a perennial woody climbing vine, black pepper achieves towering heights of 5–6 m through the extension of aerial roots along the support columns. Characterized by bright, lustrous leaves arranged in an alternate pattern, its most valuable part lies in the fruits (drupes), each measuring about 5 mm in diameter. These drupes constitute the prized peppercorns, the most valuable component of the plant. Black pepper is the dried fruit of a mature fruit (peppercorns) and is used in a variety of applications, mostly as a spice, condiment, preservative, pesticide, and herbal medicine (Wang et al. 2017).

The fiery flavor of black pepper, attributed to the presence of the pungent alkaloid piperine (De Almeida 2020), extends its influence beyond the culinary realm. Piperine is renowned for enhancing the bioavailability of various therapeutic drugs (Khajuria 2002) and stimulating digestive enzymes within the pancreas and intestines, notably enhancing biliary bile output (Tiwari and Singh 2008). Comprising trace quantities of safrol, pinene, sabinene, limonene, caryophyllene, and linalool, black pepper's global culinary ubiquity is unequivocal, used in a variety of cuisines worldwide.

The multifarious uses of black pepper transcend the realm of flavor, with documented roles in alleviating digestive disorders, stomach maladies, diarrhea, indigestion, respiratory ailments, intermittent fever and anxiety (Srinivasan 2007; Parganihaet al. 2011; Pany et al. 2016; Ghosh et al. 2021). Furthermore, it exhibits a protective shield against bacterial, insect, and animal infections (Scott et al. 2008; Ahmad et al. 2012). With a global reputation in ethnomedicine, black pepper is hailed for its diverse medicinal attributes, offering a spectrum of therapeutic benefits (Scott et al. 2008). Piper-amides extracts from black pepper display insecticidal properties while the secondary metabolite nerolidol assumes a pivotal role in mite control (Scott and Albert 2005). Pure compound ‘Piperine’ has been recognized with many more therapeutic activities such as anti-oxidant (Al-Khayri et al. 2022), anti-neoplastic/cancer (Sunila and Kuttan 2004), anti-colorectal cancer (Wu et al. 2023), anti-tumor response in breast cancer (Lasso et al. 2023), anti-pyretic (Damanhouri and Ahmad 2014), anti-apoptotic (Pathak and Khandelwal 2007), anti-hypertensive (Saleem et al. 2022), anti-platelets (Taqvi et al. 2008), anti-spasmodic (Tiwari et al. 2023), anti-metastatic (Manoharan et al. 2009), anti-mutagenic (EI-Hamas et al. 2003; Zahin et al. 2021), neuroprotective effect (Pany et al. 2016), anti-spermatogenic (Chinta and Periyasamy 2016), anti-depressant (Lee et al. 2005; Ghosh et al. 2021), anti-asthmatics (Parganiha et al. 2011), anti-thyroid (Panda and Kar 2003), free-radical scavenger (Gülçin 2005), hepatoprotective (Mushtaq et al. 2021), immune-stimulator (Pathak and Khandelwal 2009), antibacterial (Dorman and Deans 2000), anti-fungal (Zhang et al. 2021), insecticidal and larvicidal activities (Park 2012; Saleem et al. 2022). Apart from this piperine also minimizes the likelihood of arthrosclerosis through anti-atherogenic and hypolipidemic effects (Yang et al. 2019). It is purportedly used to treat gastrointestinal disorders, lung problems, fever, cold and colic disorders (Parmar et al. 1997; Ravindran 2000).

Due to the overutilization of P. nigrum, its germplasm conservation is paramount to safeguard genetic diversity and guaranteeing its continuous availability for future generations. In this review, an attempt has been made to capture the trans situ (both in situ and ex situ) methods of conservation of black pepper for future agricultural, research, or breeding purposes (Agrawal et al. 2023).

Information on propagation and conservation were drawn through the literature available on the current status of the black pepper crop in India. It included information on black pepper cultivar and varietal diversity in the states of Karnataka and Kerala. Furthermore, a survey to document on-farm conservation of black pepper in the central Western Ghats of Karnataka, India was also conducted during November 2020 to November 2021 (Fig. 1). Data on on-farm conservation of P. nigrum was documented from the different custodian farmers using a questionnaire and semi-structured interview. The data was collected through the snowball sampling technique where, custodian farmers were selected using information shared by other fellow farmers (Subedi et al. 2003). A global positioning system (GPS) was used to define and record the coordinates (latitude, longitude) of each survey area following on-farm conservation of blackpepper landraces (Rathi et al. 2019). In addition, the custodian farmers were interviewed in order to record their personal data (name, age, occupation, address, and contact information), farm details (cropping pattern, total farm area, landrace cultivation area), barriers to cultivation, community knowledge of on-farm germplasm conservation and biodiversity conservation, current and historical landrace use, and traditional uses associated with the landraces.

Fig. 1
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Map depicting the survey areas to document on-farm conservation of P. nigrum in Karnataka, India (Co-ordinates for the farmers are provided in Table 3)

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Plant propagation

Black pepper is a climber, typically propagated through various methods, including seed germination, cuttings (2–6 nodes per cutting), layering, and grafting (Ravindran 2000). However, seed propagation poses considerable challenges. The seeds exhibit a short viability window of approximately one week after harvest and are particularly susceptible to deterioration when exposed to moisture-depleted storage conditions (Atal and Banga 1962; Ravindran et al. 2000). Furthermore, seed propagation yields a small number of genetically diverse progenies due to low seed viability and high sterility in the post-fertilization phases (Hussain et al. 2011), and also seed propagation takes a long time and is troublesome (Atal and Banga 1962). Thus, traditionally, black pepper propagation predominantly relies on cuttings with 2–6 nodes, layering, and grafting for field cultivation (Abbasi et al. 2010). Cuttings are favored for their efficiency, while other propagation approaches are less prevalent due to their slow and time-intensive nature (Hussain et al. 2011; Nair and Gupta 2003).

Black pepper is commonly prone to various pathogens, such as fungi, bacteria, viruses, and mycoplasma, which pose significant challenges to conventional propagation techniques (Ravindran and Kallupurackal 2012). Internal viral and mycoplasma infections are particularly troublesome as they can be transmitted through vegetative reproduction methods (Philip et al. 1992). Consequently, the limitations of conventional propagation methods hinder the ability to meet the growing demand for planting material.

Despite its substantial economic importance, India's black pepper productivity lags behind that of other pepper-producing countries. This disparity can be related to the insufficient availability of quality planting material from high-yielding cultivars. Additionally, diseases such as foot rot and anthracnose, and insects such as the pollu beetle (Longitarsus nigripennis) further impede vegetative cultivation (Ravindran and Kallupurackal 2012). Two viruses, namely, cucumber mosaic virus and piper yellow mosaic virus are of concern as they can be transmitted to offspring plants, exacerbating the challenges faced in black pepper cultivation (Bhat et al. 2018).

Genetic diversity

The intraspecific diversity in P. nigrum might be due to the occurrence of the species in a wide range of altitudinal zones and its high adaptability to a broad range of environmental conditions. In terms of breeding behavior, black pepper predominantly relies on self-pollination (geitonogamy) due to its floral structure, sequential bloom patterns, and long pollen viability, despite some protogyny and potential for outcrossing (Krishnamoorthy and Parthasarathy 2009). While wind and insects might play a minor role, selfing with occasional inter-flower fertilization within the vine reigns supreme for this spice plant. Vegetative/clonal propagation is the most common practice for successful propagation and survival of various lines. The primary gene pool of black pepper is made up of landraces, natural mutations, improved cultivars, and even true seedlings (Joy et al. 2007; Sasikumar et al. 2007). The current cultivar diversity in P. nigrum has been evolved by accidental selection from natural hybridization and highly successful vegetative propagation, clonal selection from landraces based on economically important traits, and development of hybrids, which resultedin a great deal of varietal distinction in fruit size, shape, and fruiting behavior (Krishnamoorthy and Parthasarathy 2010).

An assessment of the genetic diversity of germplasm is essential for both its efficient utilization and conservation. Several researchers have conducted in-depth analyses to determine the genetic diversity of the Indian germplasm of P. nigrum utilizing morphological, biochemical, and molecular markers. The investigations that have been conducted based on morphological and biochemical parameters have demonstrated that black pepper genepool has diverse intraspecific variants found in both wild and cultivated populations (Mathai et al. 1981; Chandy et al. 1984; Raju et al. 1983; Kanakaswamy et al. 1985; Ravindran et al. 1997; Ravindran and Kallupurackal 2001; Kurian et al. 2002; Mathew et al. 2006; Parthasarathy et al. 2006; Zachariah and Parthasarathy 2008; Sruthi et al. 2013; Preethy et al. 2018).

Several investigations have been conducted to assess the extent of genetic variation among Indian black pepper cultivars using molecular markers such as Random Amplified Polymorphic DNA (RAPD), Amplifiable Fragment Length Polymorphism (AFLP), Inter Simple Sequence Repeats (ISSR) and Simple Sequence Repeats (SSR) (Pradeepkumar et al. 2003; George et al. 2005; Joy et al. 2007, 2011; Raghavan et al. 2010; Sen et al. 2010; Sheeja et al. 2013; Jagtap et al. 2016; Jose et al. 2017).

Considerable diversity among P. nigrum genotypes, cultivars, landraces, advanced cultivars and wild accessions has been reported (Joy et al. 2007, 2011; Raghavan et al. 2010; Sheeja et al. 2013). Using RAPD markers, P. columbrinum has been proven to be distantly related to P. nigrum and P. longum. In addition, P. nigrum landraces grown in southern and northern parts of coastal India could be distinctly segregated (Pradeepkumar et al. 2003). Distinct fingerprint profiles have been identified in the Karimunda cultivar (Joy et al. 2007) and SSR based specific bands have been identified in different black pepper accessions (Raghavan et al. 2010). Differences at the species level between two parents of Piper species including the varietal difference among various genotypes of black pepper have been identified using SSR markers (Jagtap et al. 2016).

Kumari et al. (2019) performed the first whole-genome SSR mining in black pepper and provided the results in the form of a database called PinigSSRdb (http://www.nbpgr.ernet.in:9091/index.php). From the assembled genomic sequence, a total of 69,126 SSRs were found, with one SSR for every 6.3 kb due to the SSR frequency of 158 per MB. Dinucleotides were the most common form of microsatellite repeat motif, accounting for 48.6% of the total, followed by trinucleotides (23.7%) and compound repeats (20.62%). For validation, a set of 85 SSRs was employed, and 74 of these yielded amplification products with the anticipated size. However, these putative SSR marker sites are anonymous loci (unknown chromosomal position) as they are based on 916 scaffolds rather than 26 chromosomes. Later, using ddRAD (double digest Restriction Associated DNA) based genotyping-by-sequencing (GBS) for rapid discovery of genome-wide location-specific polymorphic SSR markers (Negi et al. 2022), the first web-genomic resources, BlackP2MSATdb, was developed. This contains all 276,230 putative SSR markers found throughout the whole genome of black pepper, with an average distance of 2.76 kilobases between SSRs and a relative density of 362.88 SSRs per Mb. The black pepper reference genome shared 3176 polymorphic markers with 29 genotypes in total, of which 2015 were hypervariable. Subsequent sequencing of the chloroplast genome (Gaikwad et al. 2023) revealed that, the size of the chloroplast genome was 161,522 bp, showing a quadripartite structure consisting of 89,153 bp large single copy (LSC) area and 18,255 bp small single copy (SSC) region, which are divided by 27,057 bp long copy of inverted repeats (IRs). Furthermore, 216 SSRs were found, and 11 of these were confirmed by amplification in 12 different Piper species. These molecular investigations have laid the foundation for the effective utilization of black pepper germplasm, identification of quantitative trait loci, map-based cloning, molecular breeding through marker assisted selection, evolutionary research and further insights to the omics.

Even though there are more than 100 known cultivars, many of them are facing the risk of extinction for various reasons. These factors include the decimation of pepper crops by diseases such as foot rot and gradual decline, as well as replacement of the traditional cultivars by high yielding cultivars. The highest concentration of cultivars is found in the state of Kerala, followed by Karnataka, as indicated in Tables 1 and 2. Most of these cultivars are bisexual forms. The Western Ghats region is very high in endemic species (Nirmal Babu et al. 2015). Unfortunately, it is also among the most ecologically threatened area due to large-scale encroachments and human settlements that have proliferated over the past century. Sen et al. (2016) conducted ecological niche modeling, which revealed a shift in the niche centroid's direction and a decline in the area of suitable habitats for black pepper in the southern Western Ghats under both projected climate change scenarios for 2080. According to this scenario's analysis, trans situ conservation of P. nigrum germplasm is crucial for safe conservation of these highly valuable resources.

Table 1 Major black pepper cultivars in Kerala and Karnataka.
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Table 2 Released cultivars of black pepper in India.
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Trans situ conservation of P. nigrum: Indian efforts

Trans situ conservation represents a holistic methodology that combines diverse in situ and ex situ approaches (Dempewolf et al. 2014; Gowthami et al. 2021b; Agrawal et al. 2023). It integrates three important components: (1) in situ protection, management, and research; (2) local and regional seed and living plant collections for conservation, research, and education; and (3) national and global genebanks for plant breeding and crop research. While many nations employ complementary conservation strategies for various species, the predominant approach typically leans towards ex situ measures. In most instances, in situ and ex situ conservation efforts remain disjointed and independent. Trans situ conservation approaches transcend these limitations, facilitating the successful integration of multiple in situ and ex situ conservation, research, and education activities at the local, national, and global levels. Trans situ conservation of black pepper genetic resources in India has been accomplished via the collaboration of ICAR institutes, State Agricultural Universities, Central Universities and farming communities (Fig. 2).

Fig. 2
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Trans situ conservation of black pepper genetic resources in India

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In situ conservation

In situ conservation involves conservation of plant genetic resources (PGR) within their natural habitats, allowing these conserved species to co-evolve with the environmental changes (Maxted et al. 2001). This crucial strategy safeguards genetic resources through various means, such as biosphere reserves, forest reserves, botanical gardens, national parks, and on-farm conservation.On-farm conservation involves farmers cultivating and managing a wide range of populations, such as locally produced traditional crop cultivars, as well as associated wild and weedy species or forms, in agro-ecosystems where a crop has evolved. This approach serves to protect a wide range of traditional types that are well-suited for low-input farming and offer resilience in the face of climate change adaptation (Hodgkin and Hamilton1993). More than 50 traditional cultivars are being conserved in the on-farm by the farmers of Kerala (Mathew et al. 2006; Reshma et al. 2022). Community Agrobiodiversity Centre, Wayanad has been recognized by the Kerala State Council for Science, Technology, and Environment (KSCSTE) as a grant-in-aid institution for enhancing research and extension activities for the conservation and sustainable utilization of cultivated and wild species of pepper, including other native crops (MSSRF 2021).

To explore the practice of on-farm conservation in the context of black pepper, we conducted a survey in the central Western Ghats region of Karnataka, covering three districts viz., Uttara Kannada, Shivamogga, Dakshina Kannada. During the survey, we identified nine dedicated ‘custodian farmers’ who have been actively engaged in on-farm conservation of different cultivars/landraces spanning multiple generations. These custodian farmers are instrumental in preserving various black pepper cultivars and landraces, including but not limited to Ademane, Baalehalli, Bilemallige Sara, BiliMunda, Doddoge, Gejje Hipli, Karimallige Sara, Karimunda, Karimenasu, Kudurugunta, Kurimale, Maliyaali Gere, Malabar, Mallige sara, Motakare, Neelamundi, Okkalu, Sigandini, Thekkam BunchPepper, Thiruchugere, Uddagere, Uddakarki and Vakkalu (see Table 3 and Fig. 3 for details). Farmers revealed that they are conserving these landraces for several years over the generations for different objectives including conservation, yield parameters, quality of the pepper in terms of pungency and tolerance to pests and diseases. Additionally, farmers are also involved in exchange of these landraces with fellow farmers of the region.

Table 3 List of custodian farmers practicing on-farm conservation of black pepper landraces/ cultivars
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Fig. 3
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On-farm conservation of black pepper (P. nigrum) germplasm is being practiced by custodian farmers (A–D); Ramesh Hegde, Uttara Kannada (Sirsi), Karnataka, India from last two generations (A) AP Sadashiva, Dakshina Kannada (Puttur), Karnataka, India from 2–3 generations (B) Natesh, Shivamogga (Sagara), Karnataka from one generation (C) Vaasudeva K Naik, Uttara Kannada (Siddapur), Karnataka, India from 2–3 generations (D)

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Ex situ conservation

Ex situ conservation refers to the preservation of plant and animal species outside of their natural habitat (Panis et al. 2020). Common approaches include field genebanks (whole plants in the field, primarily for clonally propagated, recalcitrant seeded species, and forest crops), seed genebanks (orthodox seeds crops at low temperatures), in vitro genebanks (vegetatively propagated plants, species that do not produce seeds, produce recalcitrant seeds, and plants with long juvenile periods in vitro under slow-growth conditions), cryogenebanks (non-orthodox seeded species, vegetatively propagated).

Seed genebank

Plant species have been conserved by nature through inherent seeds during millions of years of evolution. Seed storage in seed genebanks at low temperature is the most convenient and often employed approach ex situ conservation strategy of seed producing crops (Hay and Sershen 2021). However, black pepper seeds are classified as ‘recalcitrant’ as seed viability decreases with reduction in moisture content below ~ 12% (Chaudhury and Chandel 1994). Hence, storage of pepper germplasm in seedbanks is not practical as they are vegetatively propagated and seeds are recalcitrant and heterozygous.

Field genebank

A field genebank is an ex situ conservation approach in which species are collected and transported to a secondary location away from their original location where they can be planted conserved as living collections in semi-isolated conditions, where natural evolution and adaptation processes are either temporarily halted or altered by introducing the specimen to an unnatural habitat with suppressed selection pressures in order for it to survive and be conserved (Panis et al. 2020). Field genebanks often have significantly more individuals per accession than botanic gardens. The primary application is for the conservation and utilization of species that exhibit the following characteristics. (a) do not produce bankable seeds (non-orthodox seeded species); (b) have extensive life cycles, making bringing up material for regular research from a seed collection problematic; (c) are generally propagated vegetatively/clonally; (d) produce few seeds, and (e) threatened and exceptional plant species (Agrawal et al. 2023). Exceptional plant species includes, species with insufficient available viable seed to preserve a minimum sustainable population of a species, species with seeds that are intolerant to desiccation at 15% RH, species with seeds that are partially desiccation tolerant but have a P50 (the time at which 50% of the seeds have died)of < 20 years at − 18/ − 20 °C, and species with seeds with very long germination times (> 1 year) or those for which germination has not yet been successful with any conventional dormancy-breaking methods (Pence et al. 2022).

Systematic efforts have been made since 1976 at the ICAR-Indian Institute of Spices Research (IISR), Calicut, Kerala, to collect indigenous germplasm of black pepper and its wild relatives. The collections span regions of Western Ghats forests from Maharashtra to Kerala including Goa, Karnataka and Tamil Nadu, the Andaman and Nicobar Islands, and the North Eastern regions of India, in addition to the key pepper growing tracts of South India (ICAR-IISR 2009). The collected germplasm has been characterized for morphological, yield, biotic, abiotic, and quality characteristics (ICAR-IISR 2022).

The ICAR-IISR established a National Repository for the exsitu conservation of black pepper germplasm (Ravindran and Nirmal Babu 1994). Because of the threat of disease and pests (for example, Phytophthora foot rot and nematodes), conservation approach at the ICAR-IISR has been four-pronged:

  • Nursery Genebank: Each accession is trailed in serial order and is continuously multiplied using the serpentine approach.

  • Clonal repository: This repository maintains 10 rooted cuttings of each accession.

  • Field Genebank: Accessions are planted for preliminary yield assessment, characterization, and evaluation.

  • In vitro Genebank: Accessions are conserved using tissue culture methods.

The ICAR-IISR Experimental Farm in Peruvannamuzhi, Kerala, houses the black pepper germplasm field genebank, and alternate field genebanks are maintained at Central Horticultural Experiment Station (CHES), Chettalli, Karnataka, and Regional Station of IISR at Chelavoor, Kerala. These repositories collectively host approximately 3466 black pepper accessions(ICAR-IISR 2022). This makes ICAR-IISR the largest repository of black pepper germplasm in the world, which includes over 1375 hybrids and 120 open-pollinated progeny (OP) lines.

Additionally, black pepper germplasm accessions are also being maintained at All India Coordinated Research Project on Spices (AICRPS) at the Pepper Research Station, Panniyur, Kerala Agricultural University (KAU), Kerala; Pepper Research Station, Sirsi, Karnataka; University of Agricultural Research, Dharwad, Karnataka; Regional Research Station, Chintapally, Acharya N G Ranga Agricultural University (ANGRAU), Andhra Pradesh; and Horticultural Research Station, Yercaud, Tamil Nadu. To provide additional safeguard to the germplasm, two alternate centers have been identified at ICAR-Central Plantation Crops Research Institute (CPCRI), Seed farm at Kidu, Karnataka and Cardamom Research Centre CRC of IISR at Appangala, Karnataka for cultivar and wild germplasm, respectively. Duplicate set of germplasm will be established in these centers in a phased manner. The information about maintenance of germplasm various AICRPS centres is presented in Table 4. ICAR-NBPGR, Regional Station, Thrissur, Kerala also maintains 98 accessions of P. nigrum in the field genebank.

Table 4 Black pepper germplasm maintained at various AICRPS centres.
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In vitro conservation

Due to the heterozygous nature of its seeds, black pepper is primarily propagated by stem cuttings. However, this method is slow, time-consuming, and susceptible to biotic and abiotic stresses in the field. To address these challenges and facilitate the multiplication and conservation of black pepper germplasm, in vitro clonal propagation protocols have been developed (Nirmal Babu et al. 1999; Tyagi et al. 1998, 2004). To achieve in vitro conservation in in vitro genebank, several researchers standardized micropropagation protocols either by direct or indirect organogenesis viz., callus and somatic embryos (Nair and Gupta 2003), shoot tips (Nazeem et al. 1992; Philip et al. 1992; Joseph et al. 1996; Nirmal Babu et al. 1997, 2007), nodal explants (Bhat et al. 1995), and leaf explants (Sujatha et al. 2003). Recently, Deepak et al. (2022) developed an effective micropropagation protocol by culturing nodal explants in MS media enriched with 0.5 mg L−1 BAP with a satisfactory rate of multiplication (15,600 plants from 12 nodal segments per year) that are easily acclimatized to the field conditions (Fig. 4).

Fig. 4
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In vitro conservation of Piper nigrum L. in In Vitro Genebank of ICAR-NBPGR, New Delhi (A–C); Stages of in vitro establishment (A) (15 days after inoculation), multiplication (B) (3 months after inoculation) and rooting (C) (4 months after inoculation), (D) Ex vitro hardening of in vitro cultures (3 week after transplanting)

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The primary goal of in vitro conservation is to reduce frequent demand for subculture, which can be accomplished in two ways: (i) maintaining cultures under ‘normal growth’ in standard culture room conditions (SCC) comprising 25 ± 2 °C temperature, 16-h light/8-h dark photoperiod and light intensity of 40 μmol m−2 s−1, and (ii) maintaining cultures under growth-limiting strategies (slow growth strategies) (Agrawal et al. 2023).At ICAR-IISR, Calicut, black pepper shoot tip cultures could be stored up to 360 days without subculture in half strength Woody Plant Medium (WPM) with 15 g/L each of sucrose and mannitol in screw capped culture tubes with 85% survival (Nirmal Babu et al. 1999). In the In Vitro Genebank at ICAR-NBPGR, New Delhi, 1,985 accessions of around 150 species belonging to six crop groups including (i) tropical fruits (449 accessions), (ii) temperate and minor tropical fruits (390 accessions), (iii) tuber crops (527 accessions), (iv) bulbous crops (178 accessions), (v) medicinal & aromatic plants (211 accessions) and (vi) spices and industrial crops (230 accessions), which include seven accessions of Piper nigrum are being conserved in vitro. In vitro cultures are being conservedon minimal media(½ MS + 0.1 mg/l IAA) for 11 months (Tyagi et al. 1998; Deepak 2022).

Cryo Genebank

In vitro conservation within in vitro gene bank faces several challenges, including the need for frequent subculturing, limitations in terms of duration, and the susceptibility of explants to endogenous bacterial contamination during in vitro clonal multiplication (Philip et al. 1992; Bhat et al. 1995; Abbasi et al. 2010; Rani and Dantu 2016). To address these limitations and ensure safe and long-term conservation, a complementary strategy is required. Cryopreservation stands out as the sole technology available for the secure, long-term conservation of plant genetic resources (Sharma et al. 2019; Panis et al. 2020; Agrawal et al. 2022a, b).

So far, cryopreservation of P. nigrum has been reported using seeds and seed-derived somatic embryos (Chaudhury and Chandel 1994; Decruse and Seeni 2003; Chaudhury and Malik 2004; Yamuna 2007; Nirmal Babu et al. 2012) (Table 5). Cryo Genebank of ICAR-NBPGR, New Delhi, is a unique multi-crop genebank, where protocol was standardized for cryopreservation of P. nigrum seeds by desiccating to 12% and 6% moisture contents, storing them in liquid nitrogen (− 196 °C), achieving survival rates of 45% and 10.5%, respectively (Chaudhury and Chandel 1994). Thereafter, the protocol was applied to cryopreserve 102 accessions of P. nigrum using seeds, except one accession which was cryopreserved using both seeds and embryonic axes (Table 6).

Table 5 Cryopreservation research in black pepper
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Table 6 Cryopreserved accessions of black pepper from Kerala, Karnataka and Tamil Nadu conserved at cry genebank at ICAR-NBPGR, New Delhi.
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Yamuna (2007) reported somatic embryos cryopreservation by the application of encapsulation-dehydration and vitrification techniques. In encapsulation-dehydration treatment, optimal post-thaw survival rate of 62% was achieved by preculturing in 0.7 M sucrose for one day, further dehydration in the laminar air flow for 6 h to attain 21% moisture content. In the vitrification procedure, the somatic embryos were precultured for 3 days on Schenk & Hildebrandt (SH) basal salt medium with 0.3 M sucrose and subjected to vitrification treatment for 60 min at 25 °C, resulting in a 71% survival rate after cryopreservation. RAPD and ISSR profiling were used to demonstrate the genetic fidelity of the conserved somatic embryos (Table 5).

However, it is important to note that in black pepper selfing with occasional outcrossing is the predominant mode of pollination (Sasikumar et al. 1992), and seed conservation only helps to conserve gene pools. To conserve genotypes in clonal crops like black pepper, it is essential to conserve clonal explants such as shoot tips and buds to maintain clonal identity and regenerate plants with higher genetic stability compared to cell suspensions, embryogenic tissue, and callus (Wang et al. 2021). Consequently, experiments were conducted at ICAR-NBPGR to standardize a cryopreservation protocol using shoot tips and nodal explants with the aim of maintaining the true-to-type characteristics of the material (Deepak 2022). However, the results were suboptimal, indicating the need for further experimentation.

Conclusion

Conservation of black pepper (Piper nigrum) germplasm is important to maintain genetic diversity and ensure its availability for future generations. Its unique flavor, culinary versatility, and diverse medicinal properties make it a cherished commodity. However, the overexploitation of P. nigrum has raised concerns about its conservation and genetic diversity. To address the challenges of conserving valuable genetic resources of P. nigrum, extensive efforts have been made in India through trans situ conservation, by integrating all realms of both ex situ and in situ conservation methods. The establishment of field genebanks, seed genebanks, in vitro gene banks, and cryo genebanks have played a pivotal role in safeguarding black pepper germplasm. Additionally, on-farm conservation practices involving custodian farmers have contributed significantly to maintaining diverse black pepper cultivars and landraces. Research over the last three decades has yielded rich dividends, and > 5,000 accessions, cultivars are being maintained in India at various labs, research stations and farmers’ fields. More research is warranted to safely duplicate this material using in vitro and cryopreservation techniques, especially for elite cultivars and unique lines that require clonal maintenance. The collaborative efforts of research institutions, universities, and farming communities in India exemplify a holistic approach to safeguarding this valuable genetic resource.