Reproductive biology of Lasiurus sindicus: a vital perennial fodder grass for arid ecosystem

Abstract

Background

In the arid conditions of Thar desert, only the plants which are adapted to the extreme conditions can grow and reproduce. Rangelands are important fodder resources which are needed to be improved for their long-term productivity and sustainability through conservation and utilization of indigenous plant species (Lasiurus sindicus, Cenchrus ciliaris, Cenchrus setigerus, etc.). In this first ever study; we investigated the reproductive features of L. sindicus, which will assist in breeding related improvement programs of L. sindicus. The findings will also enhance our understanding about the survival strategies of L. sindicus in the extreme arid conditions.

Results

Flowers of L. sindicus are of both types, staminate and bisexual with off- white colored corolla. Results of outcrossing index (OCI), pollen-to-ovule (P/O) ratio, pollen count and different pollination treatments, indicated for cross- pollination mechanism in L. sindicus. Absence of nectar secreting tissues for nectar production and fragrance, suggested for wind-mediated pollination system. Lower grain germination rate of self-pollination than that of geitonogamous pollination and open pollination, further supported the prevalence of outcrossing in the breeding system.

Conclusions

Different aspects of reproductive biology of L. sindicus, were examined which provided insight into conservation and management of this unique plant species for rangeland management programs. Floral traits, such as large pollen count, high grain setting in open pollination treatment and absence of pollinators in L. sindicus indicated towards wind-mediated out-crossing. Our findings have laid a solid foundation for various genetic studies and improvement programs of L. sindicus.

Background

Reproduction is a relatively fragile phase in the life cycle of a species for its perpetuation and represents the core of evolutionary process. The knowledge of reproductive biology of plants, including pollination biology and breeding system, is a prime requisite for elucidating the different pheno-events, which allow plant survival in harsh environmental conditions. Plant reproductive biology mainly focuses on flowering phenology, floral biology, pollen viability, stigma receptivity and pollination studies. The mode of pollination has direct impact on important aspects of gene action, genetic constitution, adaptability, genetic purity and transfer of genes.

The typical environmental conditions of Thar desert have permitted only those plants to grow and reproduce which are specially adapted to these extreme conditions [1]. In the arid ecosystem, rangelands are important fodder resources and play a crucial role in ecological balance and biodiversity maintenance. In the year 2019, the livestock population in the arid region had escalated to 56.8 million from a mere 10.34 million in 1951 [2]. The local grazing pressure is exceeding the suggested stocking rates for rangelands without due consideration. The urgent restoration of rangelands necessitates the careful implementation of grazing management practices and the adoption of suitable stocking densities [3]. Grazing, particularly with moderate to high stocking density (2–3 Adult Cattle Unit per hectare), can adversely impact soil organic carbon (SOC), its pools, and nutrient levels. However, a low stocking density (1 Adult Cattle Unit per hectare) is conducive to sustaining SOC and nutrient concentrations for maintaining the productivity in semi-arid tropical regions [4].

In India, a large area of rangelands is in degraded state and revitalization requires quality seed of forage crops. Considering these factors, there is a need to improve the long-term productivity and sustainability of rangeland ecosystems. This can be achieved through conservation of biological diversity and utilization of indigenous plant species, which are well adapted to the prevailing harsh weather conditions of desert [5].

Lasiurus sindicus Henr., a rhizomatous perennial C₄ grass species and popularly known as the “king of desert grasses” is well adapted to the arid climatic conditions. It is found in India, Pakistan, Afghanistan, Iran, Iraq, Saudi Arabia and some African countries. In India, it is confined to the north western hot arid zone. Western part of Rajasthan (regions of Jaisalmer, Bikaner and Barmer districts) is found with L. sindicus grass-dominated open pasture land, which is nearly about 6.0 mha in area [6]. Among the diverse grasslands of Thar desert, L. sindicus type occupies the largest area (80%) of land, followed by Dactyloctenium-Ochthochloa type plant community (8.64%), Aristida type (6.06%) and Sporobolus-Ochthochloa type (0.35%) [7]. In most of the arid regions, L. sindicus is usually found in association with other grass species viz., Cenchrus ciliaris, Cenchrus setigerus Vahl., Panicum antidotale Retz., Panicum turgidum Forssk. and Cymbopogon jwarancusa (Jones) Schult., as an important component for the vegetation cover.

Lasiurus sindicus has the potential to be used both for fodder production as well as for rangeland rehabilitation [8,9,10,11]. Cattle in the desert exhibit a preference for consuming L. sindicus due to its elevated nutritive value. It has good amounts of crude protein (4.6%), crude fibre (31%), neutral detergent fiber (72%), acid detergent fiber (38%), etc [6]. . Lasiurus sindicus grassland is perennial in nature (can survive even up to 20 years once well established and protected) and thrives well in the areas receiving annual rainfall below 200 mm on sandy plains, low dunes and hummocks. This warm-season grass has an ability to tolerate prolonged droughts [12].

The information on reproductive biology of L. sindicus is not available till date. Thus, in this first ever study on reproductive features of L. sindicus, we investigated the phenology, floral syndrome, pollen viability, stigma receptivity, breeding system and pollination behavior. We designed this study to sought answers to the following questions: (a) What are the reproductive features of L. sindicus? (b) What is its pollination behavior? (c) How does the reproductive system of L. sindicus affect its breeding success? Such a study will not only assist in breeding related improvement programs but also helps in unraveling the critical events occurring during the life cycle of the species. This will in turn enhances our understanding about its survival strategies and help in planning strategies for effective conservation and management.

Results and discussion

Phenology and floral morphology of L. sindicus

There are mainly two flowering seasons in L. sindicus, i.e., summer (from July to November) and winter (from February to April). In the summer season, the inflorescence emergence started in august and continued up to february in some plants with a peak in September. The life span of an individual inflorescence is 32.17 ± 8.80 days. The inflorescence is typically spike with very delicate rachis and single spikelet at every node. In each spikelet, four florets are sessile, out of which, two are staminate and rest two are bisexual. The fifth floret is pedicellate and staminate. Individual inflorescence contained 13–29 spikelets and 65–145 florets, with average of 21.49 ± 3.22 and 107.45 ± 16.12, n = 100. Number of hermaphrodite and staminate florets in an inflorescence ranged from 26 to 58 and 39–87, n = 100. All the florets are typically zygomorphic with three stamens/ anthers in staminate and one fi-bid, feathery and white stigma along with three anthers in bisexual florets. There are an average 209.72 ± 74.82 anthers and 43.78 ± 4.76 stigma in an individual inflorescence with non-significant differences in their lengths respectively.

Flowering intensity of 108.59 was observed and flowering was found proceeding in basipetal order. Floral morphological features were recorded, starting from individual inflorescence up to gynoecium (Fig. 1a to f). The floral phenology of an inflorescence can be compared at five different stages of inflorescence development. The different stages are; (a) spike within leaf sheath: the spike starts appearing but is still covered with leaf sheath, it lasts for 3–4 days (Fig. 2a to d); (b) spike outside the leaf sheath: spike with closed spikelets remains for 3–4 days (Fig. 2e to f); (c) flowering/ pre-dehiscence stage: anthers and stigma exerted from florets and lasted for about 3–4 days (Fig. 2g, l to n and s to x); (d) dehiscence stage: pollen dispersion starts from anthers, which later on turn brown and stigma turned yellowish. It lasts for about 3–4 days (Fig. 2g to i, l to o, and s to x); (e) spike near maturity: anthers and stigma began to dry out and grain formation starts (Fig. 2j, q, r, y and z); (f) mature spike: all the spikelets open up and upper spikelets began to fall (Fig. 2k). The flowering duration of a single floret was highly influenced by prevailing external environmental conditions. Maturity was faster in spikes flowering during period of high temperature and vice versa. The different floral morphological traits are detailed in Table 1.

Fig. 1

Floral parts of L. sindicus; (a) Closed inflorescence, (b) Flowering in inflorescence, (c) Closed spikelet, (d) spikelet with one open floret, (e) Androecium and (f) Gynoecium

Fig. 2

Flowering dynamics; (a-k) morphological phases of inflorescence development, (l-r) phases of stigma exertion and (s-z) phases of anther exertion

Table 1 Floral morphology of L. sindicus

Pollen viability and stigma receptivity of L. sindicus

Pollen viability increased gradually in the subsequent stages of anthesis with higher values at the fourth and fifth stages (82.40 ± 16.89% and 79.54 ± 12.82%), and decreased dramatically in sixth (58.14 ± 6.11%) and seventh (41.94 ± 7.88%) stages (Fig. 3a and Table S1). Average pollen viability recorded during different hours of the day was higher at the time of anther exertion and pollen release (10 a.m.-12 noon) and drastically decreased to 11.62 ± 0.91% at 9 a.m. on next day (Fig. 3b).

Fig. 3

Changes in pollen viability (%) and stigma receptivity (%) of L. sindicus at (a) different stages of inflorescence development (1–3 represent three pre-dehiscence stages of spike within leaf sheath, 3.6 ± 0.29 mm spike outside leaf sheath and 7.34 ± 1.67 cm spike length, respectively; 4–7 represent stages of indehisced anthers, anther dehiscence, spike near maturity and mature spike) and (b) different hours of the day

Stigmas exhibited low receptivity during pre-anthesis stages (Fig. 3a and Table S1). The highest receptivity was seen at the stage of indehiscent anthers (96.33 ± 9.83%) followed by anther dehiscence stage (92.71 ± 8.43%), while the lowest receptivity was recorded at the stage when spike is within leaf sheath (21.81 ± 2.46%). It was seen that maximum receptivity of 100 ± 10.88% was observed at 10 a.m. while the minimum receptivity of 29.20 ± 3.83% was seen at 8 a.m. and 9 a.m. (Fig. 3b).

Mating system

The average diameter of the inflorescences was 3.63 ± 0.19 mm (Table 1), which falls in the range of 2–6 mm, and thus was scored as 2 as per the criterion of Dafni [13]. Lack of temporal separation in anther dehiscence and stigma receptivity (due to synchronous maturation of male and female gametes) and same positioning level of anthers and stigma were both scored as 0. Hence, the outcrossing index (OCI) was considered 2, indicating the breeding system of L. sindicus was facultative autogamy. OCI is a general indication of the breeding system and not the final proof for it. The number of pollen grains, ovules, and P/O ratio were 121.82 ± 18.21 (Table 1), 1 and 121.82 ± 18.21, respectively. Thus the P/O ratio also indicated towards breeding system with facultative autogamy as per the criterion of Cruden [14].

Reproductive output

Grain formation in the individual spikes covered with parchment paper, and spikes of individually bagged plants, open plants, and isolated plants were observed. Wide variability in inflorescence formation and grain formation was observed among plants covered with a variety of covering materials. The results showed that among these four pollination treatments, the proportion of spikes which set grains (one-way ANOVA, F_(3,3) = 121.45, p < 0.001, Fig. 4a and Table S2) and grain formation (%) (one-way ANOVA, F_(3,3) = 1717.51, p < 0.001, Fig. 4b and Table S2) in open pollination varied significantly from the rest of the treatments. Of all four pollination treatments, open pollination had the highest percentage of spikes setting grains (100%) and grain formation (Table S2). In contrast, self-pollination had the lowest percentage of grain formation (3.55 ± 0.73%).

Fig. 4

(a) Proportion of spikes setting grains (%) and (b) grain formation (%) for L. sindicus subjected to different pollination treatments; A: Self-pollination, B: Geitonogamous pollination, C: open pollination, D: Isolation pollination

Pollen Biology of L. sindicus

Pollen counts for anthers, florets, and inflorescences were determined, showing considerable variation of 40.61 ± 6.07 (with a range of 27–60), 121.82 ± 18.21 (range of 81–179) and 11453.5 ± 2150.02 (range of 7862–15,504), n = 100. Hanging slide experiments demonstrated significant pollen dispersion at varying distances making out-crossing indispensable in L. sindicus. Pollen counts at 4 m and 8 m distances were approximately 643 and 978 pollen grains/ cm², respectively, peaking at 10 a.m. (806 and 1071 pollen grains/ cm²) and 11 a.m (754 and 1036 pollen grains/ cm²) at 4 m and 8 m distances. Thereafter, a gradual decline in the pollen count was observed. Similarly, with the modified method for pollen count estimation, slides placed in funnels at different heights of spikes exhibited maximum pollen score from the middle part of the spikes (545 pollen grains/ cm²) while the minimum pollen score from the lower part (373 pollen grains/ cm²). Anther dehiscence was influenced by environmental factors, such as temperature and humidity (Fig. 5), especially at higher temperatures.

Fig. 5

Mean monthly rainfall, RH, temperature (max.) and temperature (min.) records during two growing seasons

Compatibility system of L. sindicus

Among the four treatment combinations, inflorescences were not formed in the case where two grains from different spikes of open plants were sown together. The grain setting (%) was found lesser for self-pollination as compared to the treatment showing highest grain setting (%) of 7.35% for cross-pollination (Table 2). Therefore, as per Zapata and Arroyo [15], the self-incompatibility index (SII) of score close to 0.2 indicated towards presence of self-incompatibility of L. sindicus.

Table 2 Compatibility behavior of L. sindicus plants under different combinations

Grain germination and vigor index

The grains collected from different treatments (uncovered spikes, parchment paper covered individual spikes and spikes from white khadi cloth covered plants) conducted during 2020-21 and 2021-22 were kept for germination during November 2021 and 2022. These different grains were compared for germination (%) and vigor index (Fig. 6a and b). High germination (%) was observed for grains kept on wet filter paper in comparison to grains sown directly into the soil. Grains on filter paper started germinating on 2nd day and by the 10th day majority of the grains germinated. The grains within the soil started to germinate from the 3rd day and took about 23 days for full germination. In order to enhance clarity, grains from various treatments were compared based on germination (%) and vigor scores. Individual inflorescences covered with parchment paper demonstrated the lowest germination percentage (35.5 and 63.8) and vigor indexes (111.7 and 66.5) between the two study years.

Fig. 6

(a) Grain germination (%) and (b) Vigor index of grains collected from spikes of different treatments

Discussion

Floral syndromes, pollen viability and stigma receptivity

Floral syndromes, pollen viability, and stigma receptivity are critical to the success of pollen and pistil interactions. The objective of this investigation is to investigate these aspects in L. sindicus. Floral syndromes encompass a combination of integrated floral traits (e.g. morphology, color and scent) which indicate convergent adaptations for flowers that are pollinated by particular types of pollen carriers [16, 17]. The floral syndromes of L. sindicus (e.g., staminate and bisexual florets, zygomorphic flowers and off- white colored corolla) and lack of any nectar secretory tissue for nectar production and fragrance during our observations on floral parts along with different pollination treatments support that the pollination system of L. sindicus should be wind- mediated cross pollination.

Pollen viability and stigma receptivity play essential roles in the success of plant reproduction [18]. The existence of specific enzymes, such as esterases, peroxidases, and acid phosphatases, on the pistil indicates the readiness of the stigma [19]. Enhanced stigma receptivity on the pistil plays a pivotal role in the processes of pollination, fertilization, and the overall reproductive success of plants, ensuring the development of grain [20]. In our study, pollen viability exhibited variations during different day hours and stages of anthesis (Fig. 3a and b). The findings indicated a positive correlation between pollen viability and the different flowering stages of L. sindicus. The highest pollen viability was observed during the specific day hours when pollen grain was released, i.e. 10 a.m.-12 noon. The pollen viability gradually increased during flower development stages. The maximum values of pollen viability at anther indehiscence stage (82.40%) demonstrated that this species sustains a significant degree of sexual fertility during this phase. This phase proved to be crucial for effective pollen collection also. In our findings, we observed that stigma receptivity in L. sindicus was found to be varying at different flowering stages. Stigma receptivity was at its peak during the anther indehiscence stage (96.33%) and declined thereafter (Fig. 3a and b) with the spike nearing maturity stage. The stigma receptivity reaches minimum in a mature spike, creating optimal conditions for pollen germination and subsequent fertilization. It indicates that this species maintains a high level of sexual fertility during this specific stage. The simultaneous trend of variation in pollen viability (%) and stigma receptivity (%) at different stages of anthesis appears to be a strategic mechanism employed by this species for increasing the likelihood of successful pollination.

Breeding system

Breeding systems reflect the interaction between a plant’s internal genetic mechanisms and its external environmental conditions. To discover evolutionary traits that are shaped by genetic and ecological factors, it is necessary to understand the breeding system [21, 22].

As per Dafni [13], the value of OCI in L. sindicus was estimated to be 2, indicating that this species is having facultative autogamy. Additionally, the pollen-to-ovule ratio (P/O) of 121.82 ± 18.21 (range of 81–179), also indicated that its breeding system was facultative autogamy. But the OCI value should be considered as a general indicator of breeding systems and not as definitive proof. While the pollen-to-ovule (P/O) ratio is generally indicative of the breeding system on a broad scale, therefore it is important to consider each case within the context of its specific pollination syndrome. In recent times, there has been an increasing evidence suggesting variations in the P/O ratio from the standards of Cruden [14]. Moreover, the results of different pollination treatments indicated that there is outcrossing in L. sindicus (Table S2).

The main theme in the evolution of plant mating strategies pertains to the choice between cross-fertilization and self-fertilization [23, 24]. For most plants, the prevalent adoption of self-fertilization and outcrossing should be regarded as two potential stable outcomes in the evolution of mating systems [25, 26]. Our findings indicated that grain setting (%) in L. sindicus from open pollination, geitonogamous pollination, and isolation pollination exceeded that of self-pollination. Additionally, the grain germination rate of self-pollination was lower than that of geitonogamous pollination and open pollination, providing further support for the prevalence of outcrossing in the breeding system. Geitonogamous pollination played a secondary role in maintaining reproductive success within the breeding system, particularly when environmental conditions were less favorable for outcrossing due to changes in the environment.

Inbreeding or self-pollination is frequently observed in species characterized by small population sizes or individuals within confined habitats [22, 27, 28]. This phenomenon holds significant importance for the preservation and reproduction of such species. L. sindicus, for instance, thrives in regions with limited rainfall, subjecting it to challenging environmental conditions. The maintenance of self-incompatibility over time might be considered an evolutionary strategy, serving as a delayed mechanism for ensuring reproductive success or even a primary factor contributing to the natural setting of grains.

Conclusions

This study offers a comprehensive examination of various aspects of the reproductive biology of L. sindicus. It has provided insights into the floral biology, phenology, pollen viability and stigma receptivity. It has notably presented the novel research findings regarding the breeding behavior of L. sindicus. This species displays a range of floral traits, such as staminate, bisexual and zygomorphic flowers with off- white colored corolla, and absence of any nectar secretory tissue for nectar production and fragrance. All of these traits have evolved to facilitate wind-mediated cross-pollination. Through comprehensive investigations into outcrossing index (OCI) and pollen-to-ovule (P/O) ratio and experiments with different pollination treatments, it was determined that L. sindicus is primarily a species with out-crossing mode of reproduction. These insights into the reproductive biology of L. sindicus have implications for the conservation and management of this unique plant species. Additionally, these findings contribute valuable knowledge to the study of breeding systems and pollination ecology in various other species as well. Such insights can profoundly impact the success or failure of conservation work undertaken by biologists, although this aspect remains relatively understudied in the context of restoration research.

Materials and methods

Study species and sites

Lasiurus sindicus is an erect, tufted and branched perennial grass up to 2 m tall with linear, acuminate, convolute or flat leaves. Inflorescence is almost 8–14 cm long, erect, white, densely villous and possesses 13–23 spikelets. This species has sparse hair on leaves and bracts. The florets are both hermaphrodite and staminate types. Its seeds are covered with glumes and other appendages and the grains are yellowish to brown yellowish in color. The field work was performed at field research area (26°15’41”N, 72°59’40”E), ICAR-CAZRI, Jodhpur, Rajasthan during 2020-21 and 2021-22. The population consists of approximately 160 plants of L. sindicus grown from grains of a variety, CAZRI Sewan-1, released by ICAR-CAZRI, Jodhpur, which is maintained at the institute itself.

Observation of phenology and floral traits

To determine the different phenoevents, biology and anthesis in L. sindicus, 5 inflorescences of 20 randomly selected individuals were tagged. The observations were recorded regularly until inflorescence maturation for two consecutive years. A detailed study pertaining to number of ovule, etc. was carried out under Olympus SZX9 Stereozoom microscope and dissecting microscope. Different floral quantitative traits, viz., number of spikelets/ inflorescence, number of anthers/ floret, number of florets/ spikelet, etc., were noted with applied standard deviations in the field directly using hand lens. The duration of pollen shed, changes in stigma and anther color and stigma bending were monitored and recorded. The duration (timing from opening of the first flower up to wilting of the last flower), sequence and intensity of flowering in each inflorescence were observed. Floral morphological observations at different stages of inflorescence development were recorded. Anthesis (time of commencement, peak and termination in flowering) was observed as per Dafni [13]. Number of inflorescences was counted up to the time of first cut. Plant phenological traits like germination, first leaf emergence, flowering and fruiting period were recorded.

$$Flowering\,inensity\, =$$

$$\frac{{Average\,number\,of\,florets/\,plant}}{{Average\,number\,of\,inlorescence/\,plant}}$$

Flowering intensity was calculated as per Dafni [13].

Pollen viability and stigma receptivity

To observe the dynamic changes in pollen viability (%) on the first day of anthesis, 5 random inflorescences from 5 different plants of L. sindicus were labeled. Pollen grain was collected every hour from 6 a.m. to 6 p.m for five consecutive days. Five anthers of an inflorescence at different time interval were placed on a glass slide containing two-three drops of 2% acetocarmine solution [29] in the laboratory. The cellular condition of pollen grains was determined. Anthers were crushed to release the pollen grains into the solution, afterwards removing the anther sacs and ultimately covering the suspended pollen grains with a glass coverslip. The prepared preparation was left for 2–3 min for allowing the diffusion of dye by the pollen grains. Five different microscopic fields (10X) from each slide were selected randomly for observing the ratio of stained and total pollen grains under Leica DM 3000 compound microscope. Changes in pollen viability (%) at different developmental stages of anthesis (three different lengths of inflorescence and four different conditions of anther) were also observed. Pollen grain was collected at 10 a.m. from anthers obtained from 5 random inflorescences from 5 different labeled plants of L. sindicus and observed at five different microscopic fields (10X) (see Table S1) and (see Fig. S1).

$$Pollen\,viability\,(\% )\, =$$

$$\frac{{Number\,of\,viable\,(red\,stained)\,pollen\,grains}}{{Total\,number\,of\,observed\,pollen\,grains}}\, \times \,100\%$$

Stigmas collected from the above 5 inflorescences at different time intervals (from 6 a.m. to 6 p.m.) during the first day of anthesis and at different above mentioned developmental stages of anthesis were immersed in 3% (3 g/100 mL) hydrogen peroxide solution. The solution was dripped on cavity glass slide and continuously examined for bubbles formation. The change of reaction solution color [13] was observed under Olympus SZX9 stereozoom microscope (at 12.5X). The time taken for bubbling, number of initially formed bubbles and maximum number of bubbles/ stigma were recorded. Stigma receptivity (%) was measured as per Tong [30], by using the weights assigned for the sum of the values obtained from different observations. The observations included (a) bubbling start time; (b) bubbling rate: 3 (fast), 2 (medium) and 1 (slow); (c) bubble number: 4 (bubbles accumulated exceeding the stigma), 3 (bubbles accumulated equal to stigma), 2 (bubbles accumulated in the middle of stigma), 1 (bubbles accumulated on the stigmatic edges) and 0 (no bubble formation); (d) color change of reaction solution: 1 (blue color) and 0 (no color change). The highest total summed value of bubble densities indicated for the most receptive stigma.

Breeding system of L. sindicus

To estimate the likelihood of cross-pollination versus self-pollination, out-crossing index (OCI) and pollen-to-ovule (P/O) ratio were calculated on the basis of floral morphological traits taken under study. OCI is determined as the sum of (a) inflorescence diameter (0, 1, 2 and 3 for 0, 1–2, 2–6 and > 6 mm); (b) temporal separation of anther dehiscence and stigma receptivity (0 and 1 for presence of homogamy and protandry); and (c) herkogamy (0 and 1 for absence and presence) as per Dafni [13].

For estimating the pollen-to-Ovule (P/O) ratio, a total of 20 inflorescences from 10 different plants (2 inflorescences per plant) in anthesis phase with indehisced anthers were tagged at random. 3–5 anthers from different florets of 5 spikelets of each inflorescence were selected for counting the number of pollen grains. The selected anthers of each spikelet were used to prepare a pollen suspension by putting them in to a centrifuge tube (2 ml) containing 15% (g/ml) glucose solution followed by shaking. The pollen suspension was placed on microscopic slides and pollen grains were observed and counted under microscope at 10 different microscopic fields (10X). The total number of pollen grains/ floret (P) = the average number of pollen grains in each anther × the number of anthers / floret. The number of ovules/ floret (O) was counted by dissecting the ovary of each floret using a sharp blade, and then observing under Leica DM 3000 compound microscope and dissecting microscope. Pollen-to-Ovule (P/O) ratio was calculated as per criteria of Cruden [14].

$$Pollen\, - \,ovule\,ratio\, =$$

$$\frac{{Average\,number\,of\,pollen\,grains/\,floret\,}}{{Average\,number\,of\,ovules/\,floret\,}}$$

Controlled pollination experiments were conducted to further evaluate the breeding system of L. sindicus with four different types of treatments for two years (Table S2). The treatments included; (1) spontaneous self-pollination test: individual inflorescences were covered with parchment paper bags, N = 957 and 1330 (during 1st and 2nd year); (2) geitonogamous pollination test: individual plants of L. sindicus were bagged with different covering materials, i.e., malasiya cloth, cotton white khadi cloth and plastic clear sheet (0.3 micron thickness), N = 30; (3) open pollination test: inflorescences were tagged and got no further manipulation, N = 300; (4) isolation pollination test: individual plants were grown in isolation at different locations, N = 3. All the bags/ covering materials were removed after the inflorescences attained maturity. Grain formation (%) for each treatment was calculated after harvesting of the respective inflorescences. Grain formation (%) = Total number of grains in inflorescences which set grains/ total inflorescences harvested. The reproductive Output was calculated as per Dafni [13].

Pollen biology of L. sindicus

Pollen count from anthers of florets of 5 spikelets each of 20 inflorescences at 10 different microscopic fields (10X) under microscope was determined as per Dafni [13]. Average number of pollen grains/ floret = Pollen count/ anther × Number of anthers/ floret. Number of pollen grains/ spikelet and number of pollen grains/ inflorescence were estimated by multiplying the number of pollen grains/ floret by the number of florets/ spikelet and number of florets/ inflorescence, respectively.

Pollen dispersion up to 4 m and 8 m distance from the individual plant was determined by hanging slide experiment. In this experiment, some microscopic slides were attached to vertical wooden laths facing the nearest inflorescences (3 replicates for each inflorescence) [31]. However, to estimate the pollen dispersion from different parts (upper, middle and lower) of an individual spike, a modified technique was used. In this technique, pollen traps were constructed using petroleum jelly on microscopic slides placed in glass funnels just surrounding the inflorescences of plants of L. sindicus (4 slides in one funnel) and attached to vertical iron bars. Pollen grains were collected on hourly basis consecutively for 5 days from 10 a.m. to 4 p.m. from 5 random inflorescences. Observations were then recorded under microscope at 10 different fields (10X) without staining. Temperature and relative humidity were recorded during the pollen grain collection periods.

Detection of self-incompatibility index (SII)

The grain setting (%) of plants under 4 different treatments was recorded after flowering to calculate the SII of L. sindicus based on the method of Zapata and Arroyo [15]. Four different pairs of plants (grown by sowing grains) were covered before flowering under cotton white khadi cloth structures to evaluate for self- incompatibility: (1) sowing 2 grains from single covered plant of previous season; (2) 2 grains from 2 different covered plants of previous season; (3) 2 grains from open-pollinated (uncovered) inflorescences and (4) root slips from single plant covered in previous season. All the covered bags were removed after the inflorescences attained maturity and grain formation (%) for each treatment was calculated after harvesting of the inflorescences.

Grain germination and vigor index

The potential of the grains belonging to different treatments (spontaneous self- pollination, geitonogamous and open pollination) to germinate was tested. The grains for germination were put on moist filter paper in germination boxes under laboratory conditions and in soil containing pots for two consecutive years. Grain germination (%) = total number of germinated grains by total number of grains kept for germination. Grain vigor index I and II were also calculated.

Data analysis

Dataset with a normal distribution were analyzed using one way ANOVA. The stamen and pistil lengths were compared using Student’s t-test. One-way ANOVA was used for comparing different pollination treatments and grain setting (%). SPSS version 23.0 (IBM, USA) was used for other data analysis.