Pigeonpea [Cajanus cajan (L.) Millspaugh] holds a distinguished position among these high-protein crops, offering numerous nutritional and agronomic advantages over other pulse crops (Saxena et al., 2010). This perennial shrub belongs to the Leguminosae family, is indigenous to India and possesses a diploid chromosome number of 2n = 22 (van der Maesen, 1990).
       
A distinctive feature of pigeonpea among legumes is its unique floral morphology, which facilitates both self-pollination and cross-pollination, with varying degrees of outcrossing (5-40%) observed across different environments and locations (Saxena et al., 2016). This reproductive flexibility has significant implications for genetic diversity maintenance and breeding approaches in pigeonpea improvement programs (Bohra et al., 2017).
       
The growing demand for pigeonpea necessitates substantial increases in production capacity, requiring intensive efforts to enhance the crop’s yield potential (Saxena, 2008). Hybridization, including wide hybridization involving wild Cajanus species, is a cornerstone of these improvement efforts. Wild relatives of pigeonpea, such as C. scarabaeoides, serve as a valuable reservoir of genes for desirable traits like disease resistance and drought tolerance (Odeny et al., 2009). Among various approaches to productivity improvement, optimizing pollination efficiency represents a promising strategy (Saxena  et al., 2016). Effective pollination constitutes a fundamental prerequisite for successful fertilization and seed set in most plant species, including pigeonpea (Cajanus cajan) (Saxena  et al., 2024). To develop rational strategies for improving pigeonpea productivity through enhanced pollination, a comprehensive understanding of pollen biology is essential, including critical aspects such as pollen viability, germination capability and pollen tube growth dynamics (Dalvi et al., 2008).
       
Pollen and pollination studies help breeders identify cultivars that are compatible with each other because incompatibility has an impact on plant yield (Kamrani, 2012). The time it takes for pollen grains to remain viable after being released can vary greatly; in ideal conditions, most pollen remains viable for a few days to a week, but in dry conditions, some species of pollen are extremely vulnerable to drying out, losing their viability within an hour (Shivanna and Ram, 1993). Choudhary et al., (2011) reported that native pollen tubes have been observed to grow below the stigma relatively slowly, indicating the presence of a self-incompatibility barrier.
       
The germination of pollen and the growth of pollen tubes are preconditions for fertilization and seed development. In vitro, pollen germination is a reliable method to test the viability of pollen. Additionally, in vitro pollen tube growth is necessary to assess pollen grain germinability. In vitro, pollen germination and pollen tube growth experiments are very insightful for clarifying the absence of fertility. Therefore, a pollen germination study is needed to diagnose the crossability barriers. The most widely used basal medium for such studies is the one developed by Brewbaker and Kwack (1963), hereafter referred to as BK medium. However, the nutritional and osmotic requirements for optimal pollen germination are highly species-specific and often genotype-dependent (Soriano and Boutilier, 2013). For pigeonpea, previous studies have reported significant challenges, most notably the profuse bursting of pollen grains and tubes in standard liquid media, which necessitates specific modifications (Reddy et al., 2025).
       
These studies have received significant knowledge on the physiology and biochemistry of pollen germination and pollen tube growth. Particularly involving specific species or genera, the evaluation of pollen viability is very significant in artificial pollination. Pollen germination medium varies from species to species and even for different varieties of the crop. Linskens (1970) used basic sucrose / boric acid media and other complex media were later reported by adding polyethylene glycol and various amino acids. A commonly used medium and considered suitable for over 86 species comprising 39 plant families have been developed (BrewBaker and Kwack,1964). The cultivated pigeonpea has a variety of diverse wild species in the genus Cajanus which includes 32 species. Pigeonpea in vitro pollen germination has been previously documented and a cumulative germination rate of 48% has  been recorded (James et al., 1987 and Singh et al., 1992) which is quite low.
       
Therefore, a suitable medium for improving the pollen germination frequency and the pollen tubes’ consistency that grow in vitro must be established. This study aimed to develop an appropriate medium for the in vitro germination of pollen in cultivated and wild pigeonpea.

Cajanus cajan var ICPL 85010 which is popular cultivated species has been used to germinate pollen in present study.  Further two wild species, C. scarabaeoides from secondary genepool and C. platycapus from tertiary genepool has been used. The seed material of wild species were procured from gene bank unit of International Crops Research Institute for the Semi-Arid Tropics, Hyderabad, India during 2021. The in vitro pollen germination studies were carried out in Pre-breeding department at the International Crops Research Institute for the Semi-arid Tropics, Hyderabad, India. Pollen grain was regarded as germinated if the pollen tube’s size exceeded the pollen grain diameter (Tunistra and Wedel, 2000).
       
Two drops of media were placed on a glass slide.  Pollen grains extracted from freshly dehisced anthers was dusted on the germination medium and covered with cover slips. Pollen grains were recorded in the medium, which had germinated and produced pollen tubes. The length of the pollen tubes in the medium was measured 12 hours after germination began and the percentage of pollen germination was calculated and the average pollen tube length was recorded. Germination percentage was estimated by dividing the number of germinated pollen grains per field of view by the total number of pollens per field of view and expressed as a percentage (Kearns and Inouye, 1993). Pollen tube lengths were measured using an ocular micrometer fitted to the eyepiece of an Olympus microscope. Mean pollen tube length was calculated from each slide as the average of 10 pollen tubes’ length. For statistical analysis of data, we used one-way ANOVA analysis and Tukey’s tests (P<0.05) to compare differences between treatments.
 
Brewbaker and Kwack’s medium
 
Brewbaker and Kwack medium (Brewbaker and Kwack, 1964) was used (10% sucrose,100 mg/l, boric acid, 300  mg/l calcium nitrate, 200 mg/l magnesium sulfate and 100 mg/l potassium nitrate) as the base medium for pollen germination experiments. One hundred eight key media was developed by altering the concentration of the media constituents. The media consists of 10%, 30%, 40% sucrose, 0 ppm, 100 ppm, 250 ppm boric acid, 100 ppm and 300 ppm calcium nitrate, 100 ppm, 200 ppm and 300 ppm magnesium sulfate, 100 ppm potassium nitrate and 15% polyethylene glycol 6000. Out of which 18 (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q and R) medium shows pollen germination of 7 to 69.5%. The best three medium was then used in subsequent studies and further improved by altering sucrose concentrations. Since the liquid medium gave inconsistent results, 1% agar was added in all the media used.
       
The percentage of pollen germination was calculated by the following method
 

  

 
Statistical analysis
 
The experiments were conducted in a completely randomized design. Data were subjected to Analysis of Variance (ANOVA) and treatment means were compared using Tukey Test at a significance level of p≤0.05.

The results of studies on in vitro pollen germination of Cajanus cajan are summarized in following headings.
 
In vitro pollen germination and pollen tube growth
 
The impact of various media on pollen germination and pollen tube growth was accomplished in Cajanus cajan. Pollen germination study was examined at different concentrations of sucrose and Brewbaker and Kwack’s medium.
       
On changing sucrose concentration, boric acid, calcium nitrate and magnesium sulfate showed 7-69.5% improved pollen germination. However, pollen and pollen tube bursting was a common feature in all these media used. Pollen germinated significantly p<0.001 better in medium G compared to other media used. Pollen from medium C, H, I, L, N, P, Q and R had lower germination percentage without significant differences in germination among media (Fig 1). The agarified sucrose medium increased the germination percentage than the liquid medium. 


       
A maximum of 69.5% pollen germination was observed in a medium (G) containing 30% sucrose, 100 mg/l boric acid, 100 mg/l calcium nitrate. 100 mg/l potassium nitrate and 100 mg/l magnesium sulfate and 15% PEG. The other combinations showed lesser pollen germination and more bursting. The addition of PEG improved pollen germination.
 
Pollen tube growth
 
Pollen tubes from medium A were significantly (p<0.001) longer than those in the other mediums. Pollen from medium H, I, L, N, O and Q had shorter pollen tubes than those from other media without significant pollen tube lengths among treatments (Fig 2 and 3). 




       
The higher pollen tube length was observed in solid sucrose media than that of liquid media. A smooth, slender pollen tube was observed under solid sucrose media. Pollen tube was busted as soon as when it starts to germinate in liquid media. Similarly, budding and irregular pollen tube was also observed in case media devoid of boric acid. At higher concentration of sucrose also showed lesser germination percentage and rough pollen tube.
       
At 25^oC, the medium G (30% sucrose, 100 mg/l calcium nitrate, 100 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 100 mg/l boric acid and 15% PEG) showed the maximum pollen germination and less pollen tube bursting. The pollen of Cajanus cajan exhibited maximum germination over 69% and a mean pollen tube length of 219.7 µm in a 1% agar medium (Table 1). 


       
Hence this key medium was selected for further modification. Different combinations of the constituent’s sucrose, boric acid and calcium nitrate were essayed to enhance the pollen germination.
       
At 25^oC, the medium A2 (37% sucrose, 100 mg/l calcium nitrate, 100 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 100 mg/l boric acid and 15% PEG) showed maximum germination of 48.1 % and less pollen tube bursting with mean pollen tube length 123.4 µm (Table 2).


       
At 25^oC, the medium B1 (30% sucrose, 100 mg/l calcium nitrate, 100 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 250 mg/l boric acid and 15% PEG) showed maximum germination of 39.5% and less pollen tube bursting with mean pollen tube length 40 µm (Table 3).


     
At 25^oC, the medium B2 (37% sucrose, 100 mg/l calcium nitrate, 100 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 250 mg/l boric acid and 15% PEG) showed maximum germination of 38% and less pollen tube bursting with mean pollen tube length 49.8 µm (Table 4).


       
At 25^oC, the medium C1 (30% sucrose, 300 mg/l calcium nitrate, 200 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 100 mg/l boric acid and 15% PEG) showed maximum germination 32.5% and less pollen tube bursting with mean pollen tube length 198.5 µm (Table 5).


       
At 25^oC, the medium C2 (37% sucrose, 300 mg/l calcium nitrate, 200 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 100 mg/l boric acid and 15% PEG) showed  maximum germination of 33% and less pollen tube bursting with mean pollen tube length 59 µm (Table 6).


       
Bursting of pollen was observed at 10%, 30%, 40% sucrose solution devoid of boric acid. Pollen tubes exhibited irregularities such as coiling and bursting, in the absence of boron. Related findings were reported  by previous workers (Shivanna et al., 1997). An increase in the concentration of sucrose caused a corresponding decline in pollen germination. The addition of boric acid facilitates the pollen germination. Pollen germination was also checked at different sucrose concentrations combined with 100 and 250 ppm boric acid, germination percentage increased.
 
Pollen tube growth
 
Pollen tube length was more or less seen compared to the pollen germination level. At 30% sucrose + 100 mg/l boric acid, the pollen tube’s length was recorded to be 219.7 µm, while a 40 µm long pollen tube was observed at 30% sucrose + 250 mg/l boric acid (Table 7).


 
Standardization of pollen germination media for Wild Cajanus Species
 
The above best media was further tested in wild Cajanus viz Rhynchosia bracteata. At 25oC, the medium G (30% sucrose, 100 mg/l calcium nitrate, 100 mg/l magnesium sulfate, 100 mg/l potassium nitrate, 100 mg/l boric acid and 15% PEG) showed maximum germination 74% and less pollen tube bursting with mean pollen tube length 198.16 µm (Table 8 and Fig 4, 5 and 6).





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Brewbaker and Kwack’s medium (Brewbaker and Kwack, 1964) were used in the present study with some mild modifications for the in vitro pollen germination of pigeon pea. PEG 6000 and agar were also used to complement the pollen germination media. Good germination was obtained in agar solidified media G (Jayaprakash, 2018). This media was further tested in wild Cajanus species and found effective. Among the wild Cajanus species, Rhynchosia bracteate, a tertiary gene pool species showed good germination with more pollen tube length. Studies on in vitro pollen germination in pigeonpea (Cajanus cajan) have demonstrated that agar-solidified media promote the development of straighter and more slender pollen tubes compared to liquid media alternatives (Brewbaker and Kwack, 1963; Jayaprakash and Sarla, 2001). This morphological characteristic is significant for assessing pollen vigor and predicting potential fertilization success.
       
In pollen germinability assays conducted in vitro, sucrose commonly serves as an energy source across numerous plant species, as it typically stimulates both germination initiation and subsequent tube elongation (Taylor and Hepler, 1997). Sucrose functions not only as an energy substrate but also contributes to maintaining appropriate osmotic balance in the germination medium (Shivanna and Rangaswamy, 1992).
       
A key finding from comparative media studies is that sucrose-containing solid germination medium (agar-based) appears to be optimal for evaluating both pollen germinability and vigor (as measured by pollen tube length) in pigeonpea compared to sucrose-containing liquid media formulations (Saxena et al., 2018). This optimization of germination conditions enables more reliable assessment of pollen quality parameters, which is essential for breeding programs aimed at improving pollination efficiency and seed set in pigeonpea (Saxena et al., 2000).
       
This is in agreement with the result obtained by Stadler et al., (1999) who claimed that sucrose was the only carbohydrate that supports Arabidopsis pollen’s growth. Sucrose has the function in maintaining the osmotic pressure of the germination medium to provide enough moisture for enhance germination without rupturing pollen tubes and acting as a substrate for pollen metabolism. In conclusion, there was a significant influence of the germination medium on the pollen germinability and vigor. The liquid medium showed profuse pollen bursting due to quick hydration (Tushabe and Rosbakh, 2021).
       
Tip bursting was a common phenomenon observed in different media. Some pollen also showed budding, which is considered as ungerminated. The germination percentage tends to be decreased at higher concentration of sucrose (Shi et al., 2023). Previous liquid medium usage to germinate pigeonpea pollen has been reported for C. cajan, but only 48.7% pollen germinated (James et al., 1987). Singh et al., (1992) modified the medium by adding agar and achieved overall pollen germination of 43.1% (Singh et al., 1992; Ginoya et al., 2025). A set of media with PEG and agar were tested in the present study. In these media, germination with profuse pollen and pollen tube bursting was observed at 32.5-69.5 per cent. Medium G showed less pollen tube bursting. PEG addition has improved the germination percentage in pigeonpea. At higher concen-trations of boric acid pollen tube length was shortened (Johnston et al., 2005; Shi et al., 2023).  Another legume, chickpea, has also found it useful (Shivanna et al., 1997). Medium G showed less pollen tube bursting. This study serves as a valuable reference for breeders seeking to create variability in pigeonpea through wide crossing and developing superior cultivar with higher productivity and resistance to diseases and pests.

To meet the germination necessity of pollen, a simple or complex medium can be developed. The proposed protocol would provide better guidance in developing a pollen germination medium. The author has standardized the pollen germination medium for cultivated Cajanus cajan vars. ICPL 85010. Further, the medium was standardized in wild species, used to test the pollen germination in wild Cajanus. Besides the addition of B.K. salts, these media have been reinforced with polyethylene glycol and agar. A lot of intraspecific variabilities occurs in the entire pigeonpea gene pool. This PGM can be applied in many different ways. Intra-specific heterogeneity based on a pollen germination medium can help to select appropriate hybridization accessions.  It can aid in vitro pollination/fertilization attempts to introgress useful traits and subsequently developing suitable variety.

The authors thankfully acknowledge ICRISAT for providing the materials and research field.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

Authors do not have any conflict of interests to declare.