Ovarian Incompatibility in Tuberose (Polianthes Tuberosa L.) Cultivars and Breeding Lines

Background: Incompatibility occurs frequently in the plant system upon intra- or interspecic crosses resulting in several pollination barriers. The modest understanding on the breeding behaviour and mechanisms governing tuberose (Polianthes tuberosa L.) through this study will direct us to appraise the pollination and post pollination events on self- and cross- incompatibility and seed set behaviour for cross breeding programme. Results: Self-pollination of tuberose cultivars Arka Prajwal, Mexican Single and Arka Sugandhi failed to produce pods upon autogamy and geitonogamy. Pollen-pistil interaction study of incompatible cultivar Arka Prajwal substantiates that pollen tube enters the ovule within 24 hours after pollination. It discharges its contents into the embryo on the 1 st day after pollination and polysaccharide granules evident upon uorescence microscopic study. The pollen tube growth rates of self and controlled pollination were similar; however, sterility was expressed in the ovary. The female gametophytic development in self and cross incompatible cultivar Arka Prajwal showed normal development initially after pollination whereas embryosac degeneration was observed at later stage. Complete degeneration of the integument and nucellar cells was also observed. Conclusions: This study is the rst to elucidate the occurrence of pseudo self-incompatibility in tuberose by identifying tuberose cultivars and breeding lines which produce pods upon geitonogamy. A positive pollen-pistil interaction with the degeneration of embryosac, integument and nucellar cell and pod shrinkage, abortion and fall conrms the prevalence of the ‘Ovarian Incompatibility’ or ‘Late-Acting Incompatibility’ in tuberose.

Indian Institute of Horticultural Research, Bangalore has released six superior tuberose hybrids and working on the development of suitable varieties for processing with high concrete content. Tuberose possesses the characters of self-incompatibility and dichogamy, thereby preventing the fruit set upon sel ng (Shen et al., 1986;Seetharamu, 1993;Uma, 1990). Earlier studies on tuberose also stated that the seedlessness in tuberose, especially single owered cultivars is due to sterility (Joshi and Pantulu 1940), at present found to be owing to self-incompatibility (Shen et al., 1986 and Karihaloo, 2019).
Self-incompatibility involves the dynamic rejection of male gametophytes that contains the same Salleles as the female sporophyte (Nettancourt, 2001). This rejection stated that it happens in the stigma or style region; however, in late-acting self-incompatibility the rejection arises in the ovary (Seavey and Bawa, 1986). Late acting self-incompatibility or self-sterility involves prezygotic and post-zygotic embryo sac degeneration following self-pollination (Sage et al., 2006 andBittencourt et al., 2003). Pollen germination on stigma and pollen tube growth through the pistil and into the ovule are regulated by pollen-pistil interactions (Linskens,1986). Pollen-pistil interaction has been used to study the compatibility and incompatibility relationships in many crops including Lilium longifolium (Dickinson et al., 1982) and sweet cherry (Radunic et al., 2017).
Tuberose cultivars have also been observed to show incompatibility issues during hybridization resulting in no seed set (Hemanta, 2015). The exact cause of sterility in tuberose is not known and investigations carried out showed that sterility is not due to any defects or deformation in formation of the pollen grains and development of the embryo sac (Joshi and Pantulu, 1940). However, detailed information regarding the source and mechanism of the self and cross incompatibility within these cultivars had not been reported. There is a need to understand the mechanisms and problems associated with hybridization and incompatibility within the varieties. The crossing barrier existing thus could be identi ed through a systematic study on the interaction between the pollen and pistil of the genotypes and histological analysis. The investigation would help to identify the site of inhibition of pollens in the incompatible cross and thus methods to overcome such barriers could be developed in order to use a certain superior variety as a parent for further improvement of tuberose.

Plant material
Seven single oret type genotypes of tuberose were selected from the experimental elds of ICAR-Indian Institute of Horticultural Research (ICAR-IIHR), Bengaluru (Karnataka, India). The experimental site was located at an altitude of 930 m above MSL 12º 58' North latitude and 78º 35' East longitudes in the Eastern Dry Zone (zone 5) of Karnataka. The soil type is red loamy with the E.C. 0.26 dSm−1 and pH of 7.35. The tuberose genotypes of single type (Fig. 1) viz., Arka Nirantara, IIHR-6, Variegated, Arka Shringar, Arka Sugandhi, Arka Prajwal and Mexican Single were used for the study.

Pollination And Seed Set
Eighteen genotypes of tuberose were subjected for autogamy, geitonogamy and allogamy using 25 owers each and pod set was observed 90 days after pollination. Pollinations were carried out during July 2017 and repeated during July 2018. The owers were bagged for autogamy to observe seed set. For geitonogamy, viable pollen grains were collected from the owers on 1st day after anthesis and pollinated on the receptive stigma of owers 2 days after anthesis. Natural out crossing was observed for the allogamy. For the intercrossing the matured buds were emasculated to be used as the female parents by removing the anthers manually and bagged. Continuous crossing for two to three days was practiced observing the seed set. Cut style pollination was carried out by removing the style and pollinated just above the ovary.

Fixing And Softening Of Pistil
The pollinated pistils of crosses were collected six and 24 hours after pollination. Pistils were xed in FAA xative (70% FAA-18 ethanol: 1 formalin: 1 glacial acetic acid) for 24 hours at room temperature. Samples were prepared after 24 hours, or pistils were transferred to 70% ethanol where they can be stored for up to two months. The xed pistils were removed from the xative before sample preparation and rinsed in distilled water thoroughly; they were then transferred to 8 N NaOH solutions for 4-6 hours for softening of the tissues. Optimum softening of the pistil is required for proper staining of the sample.

Staining And Slide Preparation
The softened pistils were then rinsed in distilled water and let it stand for 24 hours to remove all the adhering chemicals prior to transfer to 0.1% (w/v) aniline blue in 0.3 M K 2 PO 4 for one hour. Aniline blue (0.1%) stain was prepared by adding 0.1 g of aniline in a beaker containing 100 ml of 0.3 M sodium phosphate (K 2 PO 4 ), stirred and transferred to amber bottles for storage. The stained pistils were then carefully mounted on glass slides using glycerin, covered with cover slips and pressed gently to keep the cover slips intact. The prepared slides were observed for the pollen tube growth and behaviour under the uorescence microscope (Leica DM LB2, Wetzlar, Germany). The pollen-pistil interaction studies were observed for the compatible crosses of tuberose.

Histological Study
A comparative histological study was done on the self and cross compatible as well as the self and cross incompatible genotypes of tuberose under controlled pollination. The pods after 1st, 2nd ,3rd ,4th ,5th ,6th ,7th ,8th ,9th and 10th day of pollination were xed in FAA (Formalin: acetic acid: alcohol) for at least 24 hours and subjected to ethanol-butanol series. The samples were subjected to para nin ltration and embedded with para n wax (60 0 C), longitudinal sections of 8µm were made and a xed on the slide using gelatin adhesive (Jensen, 1962). The sections were hydrated with alcohol series and deparra nised with xylol-butanol series and stained with haematoxyline. The sections were dehydrated and mounted with DPX and observed under microscope.

Statistical analysis
The data were analysed using SAS procedures (SAS, 1990). The variables expressed as percentages were normalized by a root square transformation. The signi cant differences between means were determined by the least signi cant difference (LSD) method at 5%.

Results
Self-pollination and fruit set Self-pollination especially autogamy between the Arka Nirantara, IIHR-6, Variegated, and Mexican Single produced pod set. Arka Shringar showed fruit set upon genitonogamy with 20.00% of fruit set which was failed to set pod upon autogamy ( Table 2). Pollen donor and type of pollination affected the fruit set percentage of Arka Shringar. The varieties under study, Arka Prajwal, Mexican Single and Arka Sugandhi had failed to produce fruits upon autogamy and geitonogamy. The percentage of pod set was noticed higher in all self-compatible varieties upon geitonogamy than the autogamy. Autogamy recorded the highest percentage of pod set in Variegated (26.19%) and in case of geitonogamy, percentage of pod set was the maximum in IIHR-6 with 80.00%.   Methods To Improve Pod Set Upon Self-pollination Studies were conducted to improve the seed set upon self-pollination and percentage of fruit set (Table  3). Cut style method + NaCl 1% recorded the highest percentage of fruit set (10.67%) than the autogamy i.e., natural self-pollination (5.58%). Number of seeds per pod (26.0), number of lled seeds per pod (21.20) and number of ill lled seeds per pod (5.00) were also found to be the highest in geitonogamy.
The other treatments namely bud pollination, bud pollination + Sucrose 2%, cut style method + boric acid 50 ppm failed to produce seed set upon self-pollination. Arti cial self-pollination (geitonogamy) was carried out in ve different stages of ower viz., 1st day of anthesis, 2nd day of anthesis, 3rd day after anthesis, 4th day after anthesis and 5th day after anthesis and fruit/seed set was observed on the 3rd day of anthesis. NaCl-Sodium chloride, NAA-1-Naphthalene Acetic Acid, GA 3 -Gibberellicacid, BAP -Benzylaminopurine

Self-incompatibility And Pollen Pistil Interaction
The pistils of the self-incompatible and compatible varieties of tuberose after 6, 24, 48 and 72 hours of sel ng was used for the uorescence microscopic study to understand the pollen pistil interaction and mechanism of incompatibility. The stigma of tuberose found re ex at maturity, wet upon receptivity and surrounded by fringe of papillae with tapering cylindrical shape protruding from the stigmatic lobes. A very few pollen grains adhered on the stigmatic surface, pollen tube growth and entry on stigma and stylar regions were limited in self-incompatible pistils of tuberose on 6 hours after pollination of Mexican Single and Arka Prajwal (Fig. 2a). Pollen tubes entered the stylar region after 6 hours of sel ng and were seen coiled in the midway after reaching 3/4th of style and further growth was arrested in some of the pistils of Mexican Single ( Fig. 2g and 2h). Lesser number of the pollen tubes was observed to reach 1/3rd of the length of the stigma at this stage as maximum of the tubes were observed to be inhibited or terminated in the upper stylar region. In contrary to that, majority of the tuberose genotypes exhibited pollen tube germination, growth and entry through the entire length of the style, base of the styles and converged into ovary region and penetrated the ovule (Fig. 2i, 2j and 2k). Pollen tube entry into the ovule via micropylar end was observed after 24 and 48 hours of self-pollination of Arka Prajwal ( Fig. 2k and 2l). The selfed pollen tube growth in the style is more active with the pollen tubes reaching the ovary within 24 hours and effecting the fertilization and after 7th and 10th day of pollination the pods shrunk after attaining pea size and abscised.

Cross Pollination And Fruit Set
Arka Prajwal, as female and pollen parent failed to set seed/fruit upon intercrossing and its reciprocally cross incompatible. The compatible cross Arka Sugandhi x IIHR-6 recorded the highest percentage of pod set (82.67%). The crossed owers of the compatible cross Arka Sugandhi with IIHR-6 as pollen donor reported highest fruit set and the pistils were collected for uorescence microscopy study to observe the pollen-pistil interaction in the normal compatible pollination. The pollens of IIHR-6 were observed to germinate abundantly in the stigma of Arka Sugandhi (Fig. 3a, 3b and 3c) and grow within the styles 6 hours after pollination ( Fig. 3d and 3e). The pollen tubes entered through the entire length of the style and nally penetrated the ovary ( Fig. 3h and 3i) and entered into the ovules (Fig. 3j, 3k and 3i) within 24 hours of pollination and leads to fertilization of the egg cells. Callose, a specialized polysaccharide which is an important cell wall component can be identi ed due to the emittance of secondary uorescence with aniline blue. The growth of the pollen tubes down the style were characterized by a series of callose plugs sealing off the pollen tube formed transversely at regular distance dividing the pollen tube into many compartments as observed in Fig. 3f and 3g. It was not found to be deposited at the tip of the growing pollen tube revealing that the pollen tube grows without inhibition within the style.

Cross Incompatibility And Pollen Pistil Interaction
The cross combinations involving the genotype Arka Prajwal as either female or male that did not resulted in pod set were taken up for uorescence studies to observe the interaction between the pollen and pistil following incompatible pollinations. The incompatible crosses with Arka Prajwal as female parent exhibited profuse pollen grains on the stigmatic surface. The pollen tubes were seen penetrating the stigma and extending into the stylar region within 6 hours, penetrated ovary and ovule upon 24 hours after pollination. The bulging of ovary was noticed initially however subsequent pod abscission was observed at pea nut stage after 7-15 days of pollination.

Post Pollination And Pod Retention
The pod retention following stigmatal pollination was observed as the number of days pods were retained on the spike after pre-treatment followed with pollination. In the cross combination Arka Prajwal x Arka Nirantara, the pod retention ranged from 5.30 days (1% NaCl) to 9.50 days (50 ppm NAA) days with a mean of 7.24 days. The maximum pod retention was observed with the stigmatal application of 50 ppm NAA (9.50 days) as compared to control (6.90 days). Among the various treatments applied in the cut style pistils, the pod retention ranges from 5.90 days (1% NaCl) to 10.30 days (50 ppm NAA) in cross between Arka Prajwal x Arka Nirantara with mean of 7.72 days. The maximum pod retention was observed in the treatment with 50 ppm NAA (10.30 days) compared to control (6.90 days). Application of different growth regulators recorded 0% fruit set and growth regulators alone helped extending the pod retention and subsequently resulted in pod abscission.

Post Pollination Events And Zygotic Barriers
The post pollination events were studied using the pods of self and cross incompatible genotype Arka Prajwal. The ovary of tuberose is observed as trilocular in Arka Prajwal and each locule contains number of ovules. The ovules were attached to the central axis of the ovary by axile placentation. The longitudinal sections of the ovules revealed that the ovules are not in same stage of development and both the mature and immature ovules are observed on the 1st day of pollination and mature ovules with the microspore mother cell and other ovules with initial stage of embryo sac formation ( Fig. 4a and 4b).
The irregular development of ovules was similar in case of self-and cross-pollinated ovules under study.
Pollen tube entry through micropylar end of the ovule and intracellular growth of the self-pollen tube between the nucellus tissue in Arka Prajwal was observed (Fig. 5a, 5b and 5c). Bitegmic ovules with organized nucellar tissue and vacuoles which later enlarges into an embryo sac were observed on 2nd day after pollination (Fig. 6a). The embryo sac containing two synergids, a central cell and an egg cell were also observed, and immature embryos showed continuous development. The 3rd day after pollination showed the matures ovules with following stages such as syngamy with the fusion of egg cell and male nuclei, zygote of resting phase and central cells without triple fusion. The observations on 4th day after pollination showed syngamy and formation of zygote (Fig. 7a). The triple fusion was also seen which later develops into endosperm which is 3n in nature ( Fig. 7b and 7c). At the 5th, 6th and 7th day after pollination the ovule has not shown any division or zygote development (Fig. 7d). The volume of embryo sac was reduced as compared to previous stage after 6th day of pollination (Fig. 8a, 8b and 8c).
Irrespective of self and cross pollination, ovule development was not seen on 8th day after pollination, in turn it showed degeneration of nutritive tissue nucellus before the formation of the embryo (Fig. 8d and 8e) which resulted in the immature pod fall and no seed set in tuberose cultivar Arka Prajwal.

Self-pollination, incompatibility and pollen-pistil interaction
Pod set observed from the cultivars upon sel ng indicates their compatibility. The results derived supports the ndings of Karihaloo (2019) and Uma and Gowda (2000) that the tuberose variety Variegated is self-compatible. Apart from that, the other single-type tuberose lines like IIHR-6, Arka Nirantara, Arka Shringar were also found to be self-compatible with the highest seed set percentage upon self-pollination. Self-pollination is restricted due to dichogamy and in the case of tuberose protandry, where the anther matures earlier than the gynoecieum which takes 2 to 3 days for receptivity. Pollen viability study indicated that some of the genotypes of tuberose continued to retain viability up to four days after dehiscence, though the percentage of viability decreased over time. The stylar polymorphism at three different levels of stigma (L-morph, M-morph and S-morph) with constant stamen is also noticed in tuberose which did not have any in uence on self-incompatibility. Species that exhibits stylar polymorphism shows ovarian self-sterility as in Narcissus triandrus (Barret et al., 2000). However, in depth research work on this aspect is required to elucidate the mechanism involved. Polianthes tuberosa with stylar polymorphism produces cymose (multi-owered) in orescence which reduces the geitonogamous self-pollination by evolution of herkogamous polymorphisms as mentioned by Barret et al. (2000) in Narcissus. The in uence of stylar polymorphism in reproductive biology of tuberose is unclear. Despite of all the differences, the results of this study provide evidence that the self-compatiblity is present in tuberose and tuberose genotypes IIHR-6, Arka Nirantara and Arka Shringar produced seed upon self-pollination. The results are in controversy with the ndings of Karihaloo (2019), Ranchana and Kannan (2016), Seetharamu (1993), and Shen et al (1987) that all the single type tuberose are selfincompatible. Among the tuberose genotypes available, few are self-fertile and most of the genotypes are self-incompatible, and some are cross-incompatible as found in sweet cherry (Choi et al., 2002). The genotype Mexican Single is self-incompatible but cross-compatible whereas Arka Prajwal is self and cross-incompatible. Self and cross pollination is essential within and among the genotypes for fruit set and for creation of variability in breeding programme.
Observation of fruit set indicated that the stigma is receptive on the 3rd day of anthesis, and early and delayed pollinations do not have any effect on overcoming the self-incompatibility. None of the methods were found to temporarily suppress the self-incompatibility in other self-incompatible genotypes of tuberose. Cut style had been successfully reported to increase pollen tube penetration (Van Creij et al., 1999 in Tulipa and Van Tuyl et al., 1982 in Lilium). However, cut style pollination failed to produce seed upon self-pollination in other genotypes of tuberose thus indicating that there is no stylar inhibition in tuberose. Bud pollination also failed to produce pods upon self-pollination with matured pollen grains and this result can be ascribed with the pistil immaturity as was observed in Easter cactus by Boyle et al. (1994).
Florescence microscopic study on the pollen-pistil interaction indicated the presence of 'gametophytic self-incompatibility' in operation as reported by Karihaloo (2019); Ranchana and Kannan (2016) in tuberose. Stylar inhibitions were also reported by Ghosh and Shivanna (1980) in Linum grandi orum, Karihaloo (2019) in tuberose and Shimizu and Okada (2000) in Arabidopsis. However, the observation of pollen tube germination, growth and entry through the style and into the ovule in other cultivars raises the possibility that negative pollen tube stylar interactions may be triggered in the late-acting self-incompatibility (LSI) species even though selfed pollen tubes continue to grow and enter the ovule for fertilization (Sage et al.1999). Slower incidence of ovule penetration in selfed pistils and consequently, a slower rate of development with the embryo sac was noted in Spathodia companulata. Moreover, in some of the cases the selfed pollen tube growth is depressed severely, and the tube never reaches the ovary (Bittencourt et al., 2003). The occurrence of embryogenic malformation, degeneration and delayed abscission of young fruits irrespective of the pollen tube entry into the ovary explains the prevalence of ovarian self-incompatibility (OSI) or late-acting self-incompatibility (LSI) in tuberose. It derives support from Uma and Gowda (2000) who stated that the pollen tube growth was uninhibited in the style con rming that the inhibiting factor was located at the ovary of tuberose. Few studies elucidates that the OSI is due to genetic control (LaDoux, 2014 and Lipow and Wyatt, 2000). In the ovarian selfincompatibility, selfed pistils are rejected though the selfed pollen tubes grew to the ovary and penetrated ovules (Bittencourt et al., 2003).

Cross pollination, incompatibility and pollen pistil interaction
Studies on pod set in several cross combinations of tuberose were also experimented by Hemanta et al. Pod abscission observed at pea nut stage might be due to the post zygotic barriers prevailing in the tuberose genotype Arka Prajwal. Arka Prajwal was used as the pollen parent showed the crossincompatibility reaction and pollen germination and pollen tube growth was not observed in the pistils of female parent except Arka Nirantara. The pollen viability and pollen germination of the cultivar, Arka Prajwal has been reported to be very low and Arka Prajwal has not produced seed set upon direct or indirect crosses thus suggesting the prevalence of sterility and incompatibility (Ranchana and Kannan, 2016). In the cross using Arka Nirantara as female parent, the pollens of Arka Prajwal were not germinated within six hours but showed slow pollen germination after 24 hours as compared to compatible cross. This showed that although the genotype Arka Nirantara showed positive interaction and selectivity to the sterile pollens as compared to the other genotypes, it showed slower germination as compared to compatible cross and eventually restriction of the pollen tubes within the style. Herrero and Dickinson (1981) had reported that incompatible pollen tubes of Petunia sp. showed slower growth as compared to the compatible one. Similar nding was also reported in Lilium by Niizeki (1959) and hazelnut by Hampson et al. (1993). The difference in the length of the pollen tube growth of direct and indirect crosses may be due to the difference in the pollen viability and germinability among the genotypes which is governed by their genetic makeup as well as the genotype speci city for a particular pollen or speci c interaction between the pollen and pistils of different genotypes. Schmidt and Timmann (1997) suggested that the pollen tube growth through the style and ovule penetration alone does not determines the cross compatibility of cultivars.

Post Pollination And Pod Retention
The increase in the number of days in cut style pollination as compared to stigmatal pollination may be due to the removal of stylar barrier as maximum of the pollen tubes were inhibited in the upper stylar region in case of stigmatal pollination. However, in case of tuberose genotypes, inspite of the increase in the days of pod retention, abortion of pods was reported before the maturation. This could be due to the post-fertilization barrier governing the genotypes which resulted in abortion. Uma (1990) had treated the incompatible crosses of tuberose with IAA, IBA and bud pollination, but recorded no fruit set, mentioning that the inhibition factor was located at the ovary after fertilization. The pollen-pistil interaction study using aniline blue staining technique was conducted to observe the post-pollination events occurring in the pistil. The interaction between the pollen and pistil in the various stages showing pollen germination, pollen tube growth and penetration within the styles and ovary were observed under the orescence microscope. The pods were retained for 8.47 days and after that the pods started changing into yellow colour, it shrunk and fell which con rms that the post pollination incompatibility in tuberose. The results derived support from the study from Shao and Wang (2020) in Jujube where regardless of self and cross pollination, the pollen tube enters into ovary and cause swelling of spherical area of the stigma.

Post Pollination Events And Zygotic Barriers
Post pollination studies revealed abnormalities in ovule development following self-and cross-pollination of Arka Prajwal. Regen (1941) in Agave virginica and Gutiérrez and Garay (2016) in tuberose also described the occurrence of unproductive ovules where embryo sac was not formed due to the degeneration of nucellar tissues. Similar observations were illustrated by Krishnamurthy and Srinivas (2005) in tuberose where the fusion nucleus did not show any signs of division whereas the symptoms of degeneration of egg apparatus in selfed ovule were observed. The results of this study indicated that the late-acting gametophytic-incompatibility exists in tuberose, where the tissue in the nucellus and the embryo sac is not supporting the zygote development and further embryogenesis is not observed (Fig. 8a, 8b, 8c and 8d). Abnormalities in the embryo sac development in tuberose variety 'Simple' was stated by Gutiérrez and Garay (2016) where three kinds of defects were seen in embryo sac development such as retarded embryo sac development, collapsed embryo sac with degradation of embryo sac and nucellar tissue, abnormal thickening of nucellar layer in the micropylar end without egg apparatus. Krishnamurthy and Srinivas (2005) furthermore described that the fusion nucleus has not gone through division upon sel ng and crossing after 1st and 4th day of pollination and exempli ed degeneration of embryo sac, egg apparatus and fruit wall on 7th day after pollination in tuberose. Uma (1990) also recorded degeneration of embryo sac in all the ovules of tuberose in shorter period after anthesis and concurrent degeneration of antipodals, egg apparatus and polar nuclei. The results derived support from Sage et al. (1999) in Narcissus where the absence of seed set might be due to the lack of vital stimulus for normal seed development upon self and cross pollination.
The current study established that the characteristic of pollination, fertilization and embryo development are associated to differences in tuberose cultivars. The recognition of self-compatible genotypes of tuberose provides a way to produce inbred lines with effective exploitation of rare resistance traits and expression of hidden recessive genetic potential. The successful fruit set is the indicator for the self and cross-compatible genotypes, despite the pollen viability, pollen pistil interaction and fertilization the ovary has the in uence on fruit set. The degeneration of embryo sac after the zygote formation was observed in the self and cross incompatible tuberose cultivar Arka Prajwal indicating that the reproductive incompatibility stimulates the metabolic activities in the associated tissues and causes disintegration of embryo sac. It is also evident that normal development of pollen pistil interaction, fertilization, double fertilization and zygote development in tuberose and abrupt degeneration of surrounding embryo sac and zygote which ultimately leads to the seed abortion in tuberose is mainly due to the self and cross incompatibility. As per Liang et al. (2005) any phase of embryo formation viz., pollen viability, pollen pistil interaction, embryo sac, zygote, nucellar tissue, embryo and endosperm might lead to seed abortion. The study on pollination, pollen pistil interaction, fertilization, zygote development and subsequent degeneration of embryo sac and zygote would be bene cial to understand the degree of compatibility for the selection of appropriate compatible parents in tuberose for the future cross breeding programme. This study elucidated the prevalence of self and cross incompatibility of tuberose genotypes and post zygotic barriers that causes late acting self-incompatibility. The current research paved the way for the detailed systematic study on the rationale of embryo sac degeneration, gene and molecular mechanism related to seed abortion in tuberose. See image above for gure legend.     See image above for gure legend.