Zygotic development and endosperm dynamics in Hevea brasiliensis: Progress, challenges and emerging opportunities of in vitro pollination and fertilisation

Zygotic development and endosperm dynamics in Hevea brasiliensis: Progress, challenges and emerging opportunities of in vitro pollination and fertilisation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Zygotic development and endosperm dynamics in Hevea brasiliensis: Progress, challenges and emerging opportunities of in vitro pollination and fertilisation Rekha Karumamkandathil, Jayasree Parukkuttiyamma Kumari, Vinoth Thomas, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7080763/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hevea brasiliensis is the primary source of natural rubber, a vital raw material with widespread applications across various industries. Owing to the consistent rise in demand for this strategic commodity, enhancing productivity remains as a key objective in rubber breeding. However, genetic recombination breeding in Hevea faces significant challenges, including seasonal flowering, low fertility, early fruit drop etc. These factors limit the production of sufficient full-sib families available for effective selection and thereby increase the cost and effort of artificial pollinations. Enhancing fruit/seed set is particularly important in a highly heterozygous perennial crop like Hevea . This study was aimed to investigate the fertilization and fruit set processes in Hevea brasiliensis , identify various factors contributing to low fruit set and explore the feasibility of an in vitro fertilization system to generate a higher number of desirable recombinants. The research was conducted using flowers and fruits from mature trees of clone RRII 105. Key factors such as age of the flower, methods of pollination, and culture media for fertilization and embryo formation were examined. Successful fertilization and embryo formation under in vitro conditions were demonstrated in H.brasiliensis . for the first time. Additionally, the study provides novel insights into embryo and endosperm development under both in vitro and in vivo conditions, shedding light on potential causes of low fruit set. The findings will be beneficial for breeders, in the recovery of hybrid seeds especially from difficult crosses of breeding programs. The protocol developed in this study can be utilized for the consistent and sustainable development of hybrid embryos into seedlings from different cross combinations. in vitro fertilization Hevea brasiliensis Endosperm development Zygotic embryogenesis Embryo rescue in vitro breeding Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Key Message The process of Pollination, fertilization , Zygotic embryogenesis, endosperm development and seed formation were studied under both in vitro and in vivo conditions in Hevea brasiliensis . Poor endosperm development was identified as a major factor contributing to the low seed set in this species. In vitro pollination experiments were successful, leading to fertilization and subsequent embryo formation. Under in vitro conditions, there is higher possibility of recovery of more number of hybrids, since the embryos without endosperm development can also be rescued. Introduction The Para rubber tree ( Hevea brasiliensis Muell. Arg.) is a commercially important species known for producing high-quality natural rubber latex, a strategic raw material with diverse industrial application (Fig. 1 ). Among the 7500 species of rubber yielding plants, H. brasiliensis is the major contributor towards the global production of natural rubber. The demand for natural rubber always shows an escalating trend and improving the productivity through developing most suitable clones with enhanced yield coupled with the prevailing biotic and abiotic stress tolerance is the need of the hour. Tree requires at least 4–5 years to attain the ripeness to blooming and retains the capacity thereafter. The recombination and selection cycles orienting towards enhanced growth and latex yield as well as the introgression of genes from wild populations to widen the genetic base through conventional crossing in rubber tree is cumbersome and laborious due to several factors like low fruit set, seasonal flowering, inaccessibility of flowers and disease infestations. High yielding rubber clones are being selected from breeding populations consists of mainly full-sib progenies, through lengthy conventional crossing followed by selection cycles (Simmonds, 1989 ; Licy et al. 2003 ; Priyadarshan and Clemet-Demang, 2004). Low fruit set, seasonal flowering, non-synchrony and inaccessibility of flowers and disease infestations are the major constraints in Hevea breeding. Low fruit set prevalent in the female parents often results in the loss of numerous potentially good cross combinations in breeding programs. Consequent reduction in the family size and recombination frequency of full-sib families results in inefficient selection, thus limiting the progress of crop improvement (Mydin et al. 1989 ). Hand pollination program is labor intensive, and the number of crosses that can be done in a season is limited due to the inaccessibility of flowers and non-synchrony (Gireesh et al. 2008 ). Despite the efforts to circumvent these problems, the fruit set could not be raised beyond five percent (Mydin 1989). On the other hand fixing of elite F1 genotypic selections through vegetative means could capture promising gene combination, which is considered as the major advantage of Hevea breeding. The presently cultivated rubber clones in Asia and Africa which contributes towards natural rubber production globally are successors of seedlings descended from Brazil to South-east Asia (Wickham collection) through Kew Garden and later undergone several cycles of breeding and selection and hence most of the present cultivars had a narrow genetic base. Reports suggests that the yield levels achievable through this currently popular Wickham gene pool is limited (James et al. 2021), warranting wide/interspecific hybridization and introgression of genes from wild populations to widen the genetic base through conventional breeding. Under such circumstances better recovery of hybrid seeds becomes inevitable and more imperative. The present study aims to examine the reasons for low fruit set and develop alternate strategies for increasing the recovery of recombinants. b. A new flush with immature inflorescence developed from the axils Flowering in Hevea is seasonal following the process of defoliation and refoliation during winter. Rubber tree is monoecious with both staminate and pistillate flowers in the same inflorescence which are developed in the axils of basal leaves of newly developed shoots (Fig. 2 a &b). Extend of flowering and seed set largely depends on the geographical location and the prevailing climatic factors. Generally flowering occurs during March-April in the northern hemisphere and September to October in the southern hemisphere (Priyadharsan 2011). In Kerala flowering follows wintering during the period from January to March. Flowering is dichogamous with incomplete protandry. Male flowers mature earlier than female flowers. The emergence of few male flowers after the maturation of female flowers makes the protandry incomplete. Female flowers are comparatively fewer and larger than male flowers and are born at the end of main branches and central axis. They are pentamerous with tricarpelary ovary (Fig. 3 ). Male flowers are numerous and smaller and seen in other parts of the panicle. The conjoined anthers often arranged in two rows over a staminal column. According to previous studies (Rao, 1961 ), Hevea is insect pollinated mainly by midges belonging to Ceratopogonidae and predominantly out crossed. immature female flowers b. Developing immature seeds separated from the ovule c. T.S of Hevea fruit with three developing seeds Mature Hevea fruit is a three lobbed capsule with a woody endocarp, a leathery mesocarp and contains three seeds (Fig. 4 a,b&c). It will take 140 days from pollination to seed dispersal (Priyadarsan, 2011). Under open pollination, among the numerous female flowers developed, only 25% form fruits and among which only 25% will reaches maturity. In controlled pollinations pollinator ensures maximum pollination, the success rate varies from 5–10 percent. In the present scenario of unprecedented climatic vagaries the availability of flowers for hand pollination itself is less. Since the peak flowering time coincides with the hot summer in Kerala which makes hand pollination difficult. The inaccessibility of flowers is another problem. Practically it is possible to do only about 70–100 pollinations per person per day on the tree top. The maximum possible pollinations that can be achieved in a season are nearly 5000–6000. That will account for around 250–300 hybrid seeds per year. In order to carryout effective selection, the family size is important. In a heterozygous species, large family size ensures maximum combinations of alleles and the best combination can be identified and selected. Thus, it is of high importance to study the fertilization event under in vitro conditions with a view to explore the possibilities of in vitro fertilization and to discuss its implications for optimizing the recovery of hybrid plantlets. The technique of in vitro pollination and fertilization is a novel approach that has been used for overcoming pre-fertilization barriers (Rangaswamy and Shivanna, 1967; Valsala et al. 1996 ) and post fertilization barriers (Sahijram, 2013). In vitro pollination and fertilization system has been accomplished in more than 57 species belonging to 14 families including Brassicacea, Caryophyllaceae, Liliaceae, Papaveraceae, Primulacea e and Solanaceae (Zentkler, 1992 and 1994). Various methods like developing interspecific and intergeneric hybrids (Zentkler, 1980; Chin et al. 1997 ), generation of haploids (Hess and Wagner, 1974 ; Musiał and Przywara, 1998 ), and pollen tube-female gametophyte interactions and the cellular events connected to fertilization and embryo development (Dupuis and Dumas 1989; Fernando et al. 1998 ) have been explored. Additionally, since the IVF system allows pollen and female gametophyte to be maintained in culture, for long periods, thus genetic transformations can be carried out on sperm or eggs coupled with hybridization (Fernando et al. 1998 ). Even though a vast literature is available on IVF systems in other plant species, in vitro fertilization studies in Hevea is meagre. A successful IVF system is relevant in a highly heterozygous perennial crop like Hevea where the breeding programmes are often delayed due to the seasonal flowering, low fruit set and early fruit drop. In this context, the present study was undertaken to elucidate the dynamics of zygote and endosperm development in Hevea brasiliensis , and to identify potential post-fertilization barriers inherent to the species. Based on insights gained from preliminary investigations (Rekha et al. 2002 ) further experiments were conducted to standardize an in vitro pollination and fertilization system for Hevea brasiliensis . Materials and Methods 1. In vitro pollination and fertilization a. Pollen pistil interaction studies Before going for In vitro fertilization experiments, we have studied the pollen pistil interaction after pollination under in vitro conditions. The fluorescent technique reported by Kho and Baer (1968) was adopted for pollen pistil interaction studies. The pollinated flowers were fixed in FAA (Johansen, 1940 ) at different intervals 12, 24 and 48 hours after pollination. After 24 hrs, the buds were transferred to 1N NaOH for eight hours at room temperature. After thorough washing with distilled water, the buds were stained in 0.1 per cent aniline blue in 0.1 N K 2 HPO 4 for period of 18 hrs. The pistil was later macerated in 90 percent glycerol. In vivo pollen tube growth was then examined under the fluorescent microscope. b. Standardization of pollination techniques in vitro Age of the flower bud The experiment was performed using the flowers of the Indian clone RRII 105. Female flowers at different developmental stages from one to three days before the anthesis (1DBA, 2 DBA, and 3 DBA), anthesis day (AD), and one day after anthesis (1 DAA) were used for in vitro pollination. Male flowers bearing pollen grains at the late uninucleate to early binucleate stage were selected for the experiment. Flower buds were collected in the early morning and washed in running tap water for three minutes. They were then sterilized using 0.1% HgCl 2 for five minutes and then thoroughly washed with sterile distilled water 4–5 times. The flowers were then blotted on a sterile filter paper to remove the moisture. Anthers were dissected from surface sterilized male flowers using fine forceps.The flowers were then blotted on a sterile filter paper to remove the moisture. Anthers were dissected from surface sterilized male flowers using fine forceps. C. Method of pollination In vitro self-pollination was done using different techniques described by Bhojwani and Razdan ( 1983 ) viz ., (i) In vitro stigmatic pollination: the female flowers after sterilization were inoculated as such in the nutrient medium. Anthers dissected from the male flowers were placed on the stigma of the female flower; (ii) Intra ovarian pollination: After surface sterilization, the perianth was completely removed, and the stigma was excised to expose the ovary. Pollination was carried out directly on the cut surface of the ovary. (iii) In vitro placental pollination: Following the careful removal of the perianth, stigma, and ovary wall, the placenta bearing the ovules was exposed. Pollen was then applied directly onto the ovules under sterile conditions. (iv) In vitro ovular pollination/test tube fertilization: The perianth, stigma and the ovary wall were removed carefully and the ovule supported by the funicle were isolated and inoculated in the medium. The anthers dissected from male flowers were placed close to the ovules. Test tube fertilization was performed as described below in four different ways. Method 1 : The ovules were inoculated in the medium and the anthers were kept aside. Method 2 : Ovules were inoculated in the medium and anthers were kept aside and few drops of sucrose solution were poured over them. Method 3 : Ovules were inoculated in the medium and pollinated with anthers and then few drops of pollen germination medium were applied over them. Method 4 : The ovules were immersed in 1% CaCl 2 for one day and pollinated with freshly isolated anthers. The pollinated flowers/ovaries/ovules were cultured on media in Petri dishes and sealed with Para film and incubated under dark conditions at 24 ± 2 o C. Unpollinated flowers/ovaries/ovules were also inoculated in the same culture medium as control. After one week, the swelled ovaries were counted as fertilized and they were transferred to new culture media in Petri dishes for embryo development and sealed with Para film and incubated under dark condition at 24 ± 2 o C. Observations were recorded at weekly intervals. 2. Culture media The basal salts of MS, WPM (Lloyd, 1980 ) and N6 (Nitsch and Nitsch, 1969 ) were experimented initially, for the growth of fertilized ovules. Based on the swelling of ovules, the basal medium was fixed and varying concentrations of sucrose from 3–18% were experimented. After fixing the sucrose level, combinations of growth regulators such as BA, KIN, GA 3 , NAA and 2, 4-D were tried for the growth of fertilized ovules. 3. Histological studies The ovules showing morphological similarities with in vivo seeds were recovered and fixed in FAA and processed for microtome sectioning. For general histology, sections were stained with Periodic Acid Schiff’s reagent (Jensen, 1962 ). For detecting starch, I 2 KI treatment and to localize lipids, Sudan IV dye (Johansen, 1940 ) was used. 4. Comparison of in vitro and in vivo fertilised ovules a. Performance of in vitro and in vivo fertilised ovules in culture In order to compare the growth of in vivo and in vitro fertilised ovules under in vitro culture conditions, in vivo pollination was performed as per standard procedure (Mydin and Saraswathiyamma, 2005) and the pollinated flowers were collected after three days and sterilized as described in the session 3.b. Ovules were isolated from these flowers and inoculated in the same medium and growth was observed. b. Growth of the fruit, endosperm and embryo under field conditions Fruits at different maturity were collected from the trees and, the developing ovules were dissected and observed for embryo/endosperm development, in order to study the endosperm growth under field conditions. The developing seeds were collected from fruits of different maturity and cultured in nutrient medium and observed for the development of embryo and endosperm. c. Growth and development of immature seeds collected from the field grown trees and cultured in vitro Open pollinated fruits from the clone RRII 105 at different stages of maturity were collected and inoculated in media following half ovulo embryo culture (Rekha et al. 2015 ). Results and Discussion 1. In vitro pollination and fertilization a. Pollen-Pistil interaction Success of pollination depends on the sequential events starting from the acceptance of pollen by the stigma and a complex and cooperative interaction between viable pollen and a receptive pistil followed by pollen tube growth through the pistil to the ovule. The pollen pistil interaction studies indicated that there is profuse development of pollen tube after pollination and it enters the ovules 24–48 hours after pollination (Fig. 5 ) in all the samples tested. These results are in conformity with earlier reports by Sedgley and Attanayake (Sedgly and Attanayake, 1986). In flowering plants, the transport of non-motile sperm to distant egg cells is required for effecting double fertilization. The pollen tube penetration of the pistil and maintenance of structural integrity of the pollen tube while moving to the destination following the positional guidance cues is required for successful fertilization event. During the penetration of pistil, the pollen tube encounters numerous physical barriers preventing the ultimate fusion between male and female gametes. Studies on pollen- pistil interactions serve as a distinctive model for elucidating the mechanisms of plant cell to cell interaction and signaling, which are critical for successful fertilization. In the present study, the sufficient growth of pollen tube ensures the absence of any pre-fertilization inhibitors in the stigma/style leading to absence of seed set. b. Age of the flower buds Among the five developmental stages assessed for in vitro pollination, the highest percentage of ovule swelling, an indicator of successful pollination, was recorded in female flowers collected one day before anthesis (1 DBA) at 79.2%, followed closely by those collected on the day of anthesis (AD) with 75.2% success (Table 1 , Fig. 6 a & b). Flowers collected one day after anthesis (1 DAA) showed a moderate success rate of 58%. In contrast, significantly lower percentages were observed in flowers collected 2 and 3 days before anthesis, indicating these stages were less responsive to in vitro pollination.This indicates that the stage of the flower is an important factor in determining the success of fertilization and it is highly specific. Immature flowers are not suitable for pollination. Fertilization success is largely determined by stigma receptivity, with the timing and duration of receptivity playing a critical role in effective pollination and subsequent seed set. It may be assumed that lack of stigma receptivity may be the reason for low fertilization success in immature flowers. In contrast, higher number of seeds was produced in Lilium when the flowers three days prior to anthesis were pollinated (Van Tuyl et al., 1991 ). Stigma receptivity is a species character and it varies among different species of same genus. In Eucalyptus, the stigma receptivity varies from 5 to 10 days among different species (Pauldasan et al 2023 ). Flowers on the day of anthesis or one or two days after anthesis improved seed set in crops like Ginger, Papaver, Nicotiana and Maize (Valsala et al. 1996 ; Kanta and Mahaswari 1963; Rangaswamy et al. 1967; Gengenbach, 1977 ). In legumes like Swainsona formosa also stigma receptivity started one day before anthesis (Zulkarnain et al. 2019 ). Even though no significant difference was noticed in the success rate of the male flowers on one day before anthesis (Fig. 6 a&b) and on the day of anthesis in H. brasiliensis , the rate of contamination was high for the flowers taken on the day of anthesis. Similarly, a high fertilization success (58%) was noticed in female flowers one day after anthesis when compared to 2 DBA and 3 DBA but the rate of contamination was high. Based on these observations, the flowers one day prior to anthesis were utilized for further experiments. Table 1 Effect of flower bud age on fertilization success after in vitro pollination in Hevea brasiliensis Treatments Age of the flower bud Success (%) 1 3 DBA 39.2 2 2 DBA 46.0 3 1 DBA 79.2 4 Anthesis day 75.2 5 One DAA 58.0 CD 9.2 DBA: days before anthesis; DAA: days after anthesis; values are means of multiple observations (p < 0.05) c. Methods of pollination The effects of different methods of pollination on fertilization success as indicated by ovary/ovule/ development are presented in the Table 2 . Based on the observations two weeks after pollination, the following results were derived. Among the four methods of pollination tried, in vitro stigmatic pollination and test tube fertilization showed some positive results, as indicated by the swelling of ovaries and ovules respectively after fertilization. The intra ovarian pollination and in vitro placental pollination failed to give any positive results and the ovules dried within a few days. Even though 40% of ovaries were enlarged under in vitro stigmatic pollination (Fig. 7 ), none of them contained seeds. In Hevea , the studies on pollen pistil interaction indicated that the pollen tubes enter the ovules 24–48 hours after pollination. This ensures the absence of any pre-fertilization inhibitions, for seed set. The fertilization may not have taken place due to some incompatibility or the embryo might have aborted at an early stage due to some post fertilization inhibitions. This observation is comparable to the available reports under in vivo conditions (Sedgley and Attanayake, 1986 ). In Hevea , there are reports that the control mechanisms for fruit wall formation and embryo growth are different and independent (Priyadharsan 2011). Hence it is possible that fruit wall may grow even after embryo abortion. Lack of some essential nutritional factors can also be attributed to the absence of seeds in in vitro stigmatic pollination of Hevea . Development of fruits after stigmatic, stylar and intra ovarian pollination without seeds is reported in the in vitro fertilization of ginger (Valsalaet al. 1996 ). In contrast, the application of pistil culture and in vitro stigmatic pollination techniques significantly improved seed set in Petunia and (Rangaswamy et al. 1967; Usha, 1965 ). Table 2 Effect of pollination method on in vitro pollination of Hevea brasiliensis Method of pollination Cultures with ovary development (%) Cultures with ovule development (%) Stigmatic 40 0.0 In vitro placental 0.0 0.0 Intra-ovarian 0.0 0.0 Test tube -- 80 Among the different methods tried for test tube fertilisation, immersing the ovules in 1% CaCl2 for 24 hrs followed by pollination and pouring pollen germination solution over the pollinated ovules was promising. 80% of the pollinated ovules swelled. Inoculating the ovules in the medium and keeping anthers over them didn’t work. Pouring sucrose solution after pollination resulted in the swelling of 10–12 percent ovules. However these ovules dried after a few days. d. Growth and development of ovules after test tube fertilization In the current study, after in vitro ovular pollination (test tube fertilization), about 80% of the ovules enlarged two weeks after pollination. The fertilized ovules were ivory colored and opaque, whereas the unfertilized ovules were glassy in appearance, turned brown and dried after a few days (Fig. 8 a). The unpollinated controls also turned brown within a few days. The swelled ovules were further cultured in different media combinations. Among the swelled ovules, further development was noticed in 40% of ovules. The rest of the ovules dried off after one month. Among the remaining swelled ovules, two types of developments were noticed wherein the first 10% ovules as a whole developed into small seeds. At 30 days post-fertilization, the in vitro-derived ovule attained a size similar to its in vivo counterpart, although its morphological characteristics closely resembled those of a mature seed. After 30 days, the size of the in vitro raised fertilized ovule was comparable with in vivo ovule but the morphology of the in vitro developing ovule resembled a mature seed under field conditions. The seed coat is sclerified and characteristic mottling was observed in the surface of these ovules as in the case of mature seeds (Fig. 8 .b). Since further embryo growth could not be observed in these cultures, they were fixed for anatomical studies. The remaining 30% of the ovules showed a different pattern of development. The embryos were developed without endosperm and seed coat formation (Fig. 8 c). By approximately 30 days after pollination, the integuments were observed to be displaced, exposing embryos at the globular stage. This is in contrast to the development of seeds under in vivo conditions. The lack of endosperm formation or its early degradation during the zygote or young embryo stage has been documented in several plant species, often posing a major constraint to successful embryo development (Kapoor, 1959 ; Töpfer and Steinbiss, 1985 ; Zenkteler and Nitsche, 1985). In the interspecific crosses of brassicas also absence of endosperm is observed after in vitro pollination. However, these embryos could be developed in to plantlets. The absence of endosperm may be either due to the failure of triple fusion during fertilization or due to endosperm abortion due to embryo endosperm incompatibility. Otherwise, after fusion, triploid nuclei may be degenerated due to some unknown reasons. The lack of proper nutrition can also contribute to the absence of endosperm. Another possible reason may be the suppression of the triploid endosperm by the faster growth of the diploid embryo. The development of embryo and germination of seedlings from the embryos without endosperm formation after IVF has been reported in Chicory (Castano, 2000). Similarly, in the test tube fertilization of Eschscholzia californica the embryo showed normal development during its early growth whereas the endosperm was purely developed and degenerated. This indicates the possibility of recovery of seedlings from such embryos in Hevea also. Optimization of nutritional requirements will be worth trying for saving these embryos. A few embryos could be recovered from these cultures and they were grown up to the cotyledonary stage. However, the size of the embryos was very small. e. Histological observations The fertilized ovules, with seed coat development were subjected to anatomical studies in order to understand the development pattern. The anatomical studies of the ovules 90 days after pollination revealed the presence of two layered outer walls (Fig. 9 .a). Well-developed seed coat with a thin walled inner zone with radially elongated sclerified cells could be seen at this stage. Seed coat is single layered in the chalazal region. The abundance of starch and lipids were observed in the nucellar area (Fig. 9 .b. & Fig. 9 c). The zygote and primary endosperm nuclei at single cell stage were also visible. This indicates successful fertilization after in vitro pollination. When an ovule of the same age after in vivo pollination was observed, the seed coat was soft and ivory in color and not similar to in vitro in the case of seed coat. Under in vitro conditions, the seed coat formation seems to be faster and has been almost completed in about 90 days. The hard seed coat may be preventing the absorption of nutrients from the medium which may in turn effect further development of the embryo. Subsequent experiments will focus on the removal of integuments at defined intervals following pollination. The pollination success as indicated by the swelling of ovules under both in vivo/ in vitro conditions is presented in Table 3 . In the in vivo pollination followed by in vitro culture, an initial success of 67.5% based on the swelling of ovules was achieved after 2 weeks of culture. Among these, only 19 percent of swollen ovules showed continued growth and rest of them dried off. In the case of in vitro fertilised ovules, among the 80% of swelled ovules 41 percent showed sustained growth after 1 month. Subsequently, they showed two types of growth pattern as described earlier. A few embryos in both in vitro and in vivo conditions developed up to cotyledonary stage (Fig. 10 ). Even though the initial success is comparable, more ovules showed sustained growth after IVF, compared to in vivo fertilization. After initial swelling of the ovules, the possibility of early embryo abortion cannot be ruled out in this situation. The sudden stress induced due to the detachment of pollinated flowers from the tree and the sterilization process might have triggered early embryo abortion. The in vitro-fertilized embryos were smaller in size compared to the in vivo-fertilized embryos. Table 3 Pollination success under in vivo and in vitro pollination as indicated by the swelling of ovules Methods of pollination Percentage of ovules swelled After 14 days After 30 days In vivo pollination 67.5 19 Test tube fertilization 79.9 41 T stat 20.6 34.79 e. Culture medium The success of in vitro pollination depends on the formulation of appropriate medium, which will support pollen germination and tube growth leading to fertilization and zygote development. MS basal salts supplemented with 100 mg L -1 boric acid and 20 gL -1 sucrose promoted pollen germination and effective fertilization, as indicated by swelling of ovules. Among various basal media and concentrations of sucrose tried out, more number of swelled ovules were obtained in MS medium supplemented with 50 gL -1 sucrose. The influence of sucrose on embryo development is well studied in other crops. In Prunus , in vitro pollination followed by culture on a medium containing 15% sucrose resulted in a high frequency of embryo development. (Dziedzic et al. 1999 ). In the media, for ovule growth, organic supplements viz. , CW (20%), malt extract (200 mg/l), casein hydrolysate (400 mg/l) and banana powder (100 mg/l) was added as it was found to be good for the rescue of embryos from immature fruits in Hevea (Rekha et al. 2010 ). Among the five growth regulators tested, Kinetin (2mgL -1) and GA 3 ( 2mgL -1 ) were found to exhibit positive results when supplied individually. Other growth regulators such as 2, 4-D, NAA and BA supplemented in the medium, resulted in callus induction from the outer integument and prevented further development of the ovule. However, when kinetin (2.0 mg L -1 ) was combined with 2mgL -1 GA3, the growth of the fertilized ovule was improved. A few embryos could be grown up to cotyledonary stage (Fig. 10 ). Further increase in concentrations of both had an adverse effect on ovule development. Addition of Kinetin promoted ovule development after in vitro pollination of two different species in Brassicas (Sosnowska and Teresa Cegielska-Taras, 2014). Kinetin had a positive effect on the growth of immature embryos rescued from field in H. brasiliensis. Experiments are underway to improve the nutritional status of the medium, for achieving further growth of the embryo. Standardization of the media requirements is very difficult using IVF embryos since tangible number of embryos could not be recovered with the present success rate. Hence experiments are being performed using open pollinated fruits. In Hevea , embryo culture was successful from 8 week old fruits onwards. Experiments are underway for the media optimization and culture requirements for very immature fruits (1–8 weeks old). Once this is achieved, the same can be used for IVF embryos as well. f. Endosperm development under in vitro and in vivo conditions When different stages of fruits (1–5 months) were dissected, it was observed that the embryos are visible only in later stages of fruit development (4–5 months). The different stages of fruit development (2–12 weeks) with their corresponding seeds are given in the Fig. 11. It is observed that, in Hevea , the growth of the fruits and seeds follow a specific pattern. The fruit wall grows faster and by 2–3 months it becomes thickened. After thickening of the fruit wall, the seeds will starts developing and when it fills the locules, the endosperm development initiates. After the completion of endosperm development, the embryo starts developing. By this time, the fruits will attain maximum growth, seed coat will be thickened and the endosperm will be fully developed. Once the development is initiated, the embryos grow faster and a fully developed embryo with cotyledons intruding in to the endosperm will be formed within 10–15 days. Different stages of fruits 2–10 weeks old) with their corresponding ovules are given in Fig. 11a. No embryos are visible in any of these stages. Fruits of 15–18 weeks maturity are shown in Fig. 11b. In H.brasiliensis , it is reported that, under field conditions, the embryos will be small and microscopic till 14 weeks after fertilization (Muzick, 1954).Our results are in conformity with this. Only during the last few weeks of fruit maturity we could see the embryos (Fig. 12 ). When mature seeds developed in vivo are observed, many seeds are without endosperm development (Fig. 12 a). In this case, embryos also seemed to be degenerated. Partially developed endosperm was also observed in some seeds where the embryos are live and fully developed (Fig. 12 b &c).In immature fruit culture, it was observed that endosperm development is partial or completely absent in many of the ovules. However, embryos will grow normally in culture even in the absence of endosperm (Fig. 12 .d) and plantlets could be recovered from these embryos. These observations reveal that embryo- endosperm communication is apparent under field conditions and may be attributed to the low seed set. After double fertilization, seed development initiates with the endosperm formation from the primary endosperm nucleus and development of embryo from the zygote. It is assumed that the development of endosperm and embryo is happening in coordination under in vivo condition. Although embryo and endosperm development are distinct processes, proper seed formation depends on elaborate and synchronized communication between the two. There are accumulating evidences which demonstrates the requirement of certain protein regulators originating from the endosperm for proper development of the embryo (Ingram et al. 2020). The ratio of maternal to paternal gene products in the endosperm plays a critical role in determining the success of crosses between individuals of differing ploidy levels within a species, as well as interspecific crosses. Maternally expressed small-interfering RNAs (siRNAs) are believed to regulate this balance by targeting growth-promoting genes, thereby influencing endosperm development and hybrid viability (David Haig, 2013 ). The endosperm acts as a major metabolic sink for absorbing nutrients from the maternal tissues and sequestering them in the central vacuole. Nutrients are then re-exported to the embryo from the endosperm (Ingram et al. 2020). In Hevea , early fruit drop is common under natural conditions as well as after hand pollination. These observations reveal that embryo-endosperm communication is apparent under field conditions and may be attributed to the low seed set. In the present experiment, after in vitro fertilization a few embryos developed without endosperm development. When mature fruits from the field are observed, there also endosperm is absent/partially developed. These observations lead to the assumption that failure of endosperm may be one of the main reasons for low fruit set. Hence future investigations focusing on these aspects need to be carried out. In immature fruit culture, the embryos developed with /without endosperm but plant regeneration was successful from both cases. Embryo rescue and culture is most successful in the recovery of plants where fruit drop is caused due to endosperm degradation. It is generally presumed that the tissue culture medium compensates for the absence of endosperm by supplying the essential nutrients required for embryo development.Continued refinements in culture media have permitted younger and smaller embryos to be rescued and grown to maturity in many crops (Stewart, 1981 ). In our experiments also, embryos could be rescued and successfully regenerated in to plantlets. Under in vitr o conditions, the embryo/endosperm dependency is not visible and many of the embryos develop into a seedling surpassing the endosperm influence. This opens up the possibility of recovering more number of hybrid seedlings by in vitro pollination. In Hevea , early fruit drop is a common phenomenon under natural conditions as well as during artificial pollination. Comparable embryo development was observed when fertilized ovules derived from in vivo pollination were subsequently cultured under identical conditions. In Spite of high initial success (67%), the value was substantially reduced (17%) (Table.3).This may be attributed to Lack of proper communication between endosperm and embryo leading to the degeneration of either of them. However, further research is needed to confirm the hypothesis. Deriving accurate nutrient composition for promoting the independent growth of the zygote is a major challenge in the success of in vitro fertilization and plant recovery. There is a possibility of inducing calli/secondary embryos from the zygotic embryo and uniform seedlings can be generated. Induction of zygotic polyembryoni and development of uniform seedlings are already reported in Hevea , using open pollinated immature seeds (Rekha et al.2015). This possibility can be applied to IVF embryos as well and multiple seedlings of desirable crosses can be generated. They enables replicated evaluation in the nursery stage itself and selection will be more effective. This is especially important in the case of perennial tree crop like Hevea , where long term field trials are inevitable for a meaningful selection of elite individuals. Callus induction and subsequent embryogenesis enables development of elite, uniform rootstocks. Embryogenic calli derived from the zygote is an ideal explants for gene transfer experiments, since the regeneration and hardening will be more efficient. There is also a possibility of developing triploids of desired combinations. In a triploid, 2 sets of maternal chromosomes and one set of paternal chromosomes will be there. Only for compatible crosses, endosperm develops in vitro . Hence there is always a possibility of regenerating viable triploid plants through somatic embryogenesis. Conclusions The present study explores the feasibility of achieving in vitro pollination and fertilization in Hevea brasiliensis , followed by normal embryo development under controlled conditions. The results confirm successful fertilization and embryo formation, with ovules cultured up to 90 days post-fertilization, leading to embryo development up to the cotyledonary stage. Further refinements in pollination techniques, along with optimization of culture media composition and environmental conditions, are likely to enhance embryo maturation and facilitate subsequent seedling development. This approach could significantly improve the recovery rate of hybrid seedlings, thereby increasing the efficiency of selection for elite traits in breeding programs. The study also highlights that defective endosperm development or impaired endosperm-embryo communication may contribute to low fruit set under natural field conditions a limitation that is circumvented under in vitro conditions, allowing for higher plant recovery. Beyond enhancing hybrid production, a successful in vitro fertilization system offers a platform for advanced crop improvement strategies, including gene transfer, gene editing, and the development of uniform elite rootstocks. Abbreviations RRII Rubber Research Institute of India IVF In vitro fertilization DBA Days Before Anthesis DAA Days After Anthesis AD Anthesis Day FAA Formalin Acetic acid MS Murashigae and Skoog WPM Woody Plant Medium SiRNAs Small-interfering RNAs BA Benzyl adenine GA Gibberellic acid NAA Naphthalene Acetic Acid 2, 4, D 2, 4, Dichlorophenoxy acetic acid FAA Formaline Acetic Acid CW Coconut Water CH Casein Hydrolysate Declarations ACKNOWLEDGEMENT We express our sincere thanks to Dr. James Jacob, former Director of Research of the Rubber Research Institute of India for the encouragement of this study. This work was supported by the Rubber Board of India. Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Competing Interests The authors have no relevant financial or non-financial interests to disclose. Conflicts of interest The authors declare no conflict of interest. Author contributions All the authors contributed equally to the work. The study was conceptualised and designed by the senior authors Rekha K and Kumari Jayasree. Material preparation was done by Raheena KT and Athira.P.V , data collection and analysis were performed by Vinoth Thomas, GireeshT ,Jithu George and Alex Raju . The first draft of the manuscript was written by Rekha K and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability statement No additional data is associated with this manuscript References Bhojwani SS, Razdan MK (1983) Plant Tissue Culture: Theory and Practice. 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Environ Exp Bot 21(3–4):301–315 Supplementary Files Graphicalabstract.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7080763","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":505147802,"identity":"5cc1fbe6-d37c-495e-b8a3-c192109db4ad","order_by":0,"name":"Rekha Karumamkandathil","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYFACNjYGhgoJOTb5wwcfALk8fMRpOWNjzC/BlmwA0sJGlBbGtrTEmTN4zCTAfEIazNvb0h78YDvMuOF2g1nl1xw7GTYG5oePbuDRInPm2HHDHp7DzAZ3DqTdlt2WDHQYm7FxDh4tEhLpbRI8EofZDA4kHLstuY0ZqIWHTRqvFvnnbZJ/DA7zGBxIbCuW3FZPhBYJtmPSPAlpEpIzktkYP247TIQWnrQ0aZkDNgb8PMeYpRm3HedhYybkF/ZjZpJv/0nUt7H3f/z4c1u1PT9788PH+LSgAGYeMEmschBg/EGK6lEwCkbBKBgxAACQk0RiNA3PHwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-2502-1583","institution":"Rubber Research Institute of India","correspondingAuthor":true,"prefix":"","firstName":"Rekha","middleName":"","lastName":"Karumamkandathil","suffix":""},{"id":505147803,"identity":"e346b26c-4674-46d3-a67c-2c5c0d4a1f43","order_by":1,"name":"Jayasree Parukkuttiyamma Kumari","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Jayasree","middleName":"Parukkuttiyamma","lastName":"Kumari","suffix":""},{"id":505147804,"identity":"e0bd691a-59dc-438e-8ea5-8f4ffcad441b","order_by":2,"name":"Vinoth Thomas","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Vinoth","middleName":"","lastName":"Thomas","suffix":""},{"id":505147805,"identity":"7fe8c017-d269-4bee-9bfa-7d17ace24376","order_by":3,"name":"Gireesh T","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Gireesh","middleName":"","lastName":"T","suffix":""},{"id":505147806,"identity":"fe359470-de67-4285-bf0f-615e25a9d6c3","order_by":4,"name":"Raheena K T","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Raheena","middleName":"K","lastName":"T","suffix":""},{"id":505147807,"identity":"cdc77135-a15f-4d12-bb12-89301ed9994f","order_by":5,"name":"Athira P V","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Athira","middleName":"P","lastName":"V","suffix":""},{"id":505147808,"identity":"5c609924-9ffb-4772-a6ce-d5d53820b0d5","order_by":6,"name":"Jithu George","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Jithu","middleName":"","lastName":"George","suffix":""},{"id":505147809,"identity":"393711d7-94ec-4f22-9d60-083ca2e00106","order_by":7,"name":"Alex Raju","email":"","orcid":"","institution":"Rubber Research Institute of India","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Raju","suffix":""}],"badges":[],"createdAt":"2025-07-09 06:57:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7080763/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7080763/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90416083,"identity":"0b8006ab-d56c-47fa-8d98-f3b1498ef6c7","added_by":"auto","created_at":"2025-09-02 13:14:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":292573,"visible":true,"origin":"","legend":"\u003cp\u003eA mature rubber plantation where plants are being tapped for the collection of latex, which is the main source of natural rubber\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/11f632629a7d5e3ccb2e152a.jpg"},{"id":90416347,"identity":"2087fd03-c2c9-4221-8c5d-5087fe80ee3c","added_by":"auto","created_at":"2025-09-02 13:22:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":542081,"visible":true,"origin":"","legend":"\u003cp\u003ea. A newly emerging shoot with initiation of flowering\u003c/p\u003e\n\u003cp\u003eb. A new flush with immature inflorescence developed from the axils\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/6e897753065f941f5c05cb58.png"},{"id":90416348,"identity":"4e450cf6-75dd-4438-9764-06d90c4d42f4","added_by":"auto","created_at":"2025-09-02 13:22:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174023,"visible":true,"origin":"","legend":"\u003cp\u003eA protandrous inflorescence of rubber tree with matured male flowers and\u003c/p\u003e\n\u003cp\u003eimmature female flowers\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/f78136f53b9f9a1ac49a0bc4.jpg"},{"id":90417540,"identity":"1434513f-49cd-4bbd-a208-f8e2e55b47db","added_by":"auto","created_at":"2025-09-02 13:30:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":511616,"visible":true,"origin":"","legend":"\u003cp\u003ea. Fruit of \u003cem\u003eH.brasiliensis\u003c/em\u003e with 3 lobes\u003c/p\u003e\n\u003cp\u003eb. Developing immature seeds separated from the ovule\u003c/p\u003e\n\u003cp\u003ec. T.S of \u003cem\u003eHevea\u003c/em\u003efruit with three developing seeds\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/1a89361e9d9065d6d7626fe4.png"},{"id":90416085,"identity":"b3cd3f2b-22c0-455b-bd26-d0eacc74ced7","added_by":"auto","created_at":"2025-09-02 13:14:55","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":36201,"visible":true,"origin":"","legend":"\u003cp\u003eProfuse growth of the pollen tube and penetration of the ovules (x100)\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/9fe465086bacebe2f91b7b41.jpg"},{"id":90417538,"identity":"f5588db7-48e2-4e36-b5c2-4f36ea7de5c5","added_by":"auto","created_at":"2025-09-02 13:30:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":557506,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Male flowers;\u003cstrong\u003e b.\u003c/strong\u003eAnthercolum with pollen at late uninucleate to binucleatestag\u003c/p\u003e\n\u003cp\u003ec. Female flowers d .Excised ovules\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/6f50211e67e1e5083036180c.png"},{"id":90418759,"identity":"9d593821-907e-426c-ad1c-1732b3e1e1a1","added_by":"auto","created_at":"2025-09-02 13:39:04","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":24894,"visible":true,"origin":"","legend":"\u003cp\u003eA swelled ovary 2 weeks after performing \u003cem\u003ein vitro\u003c/em\u003e stigmatic pollination\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/e57a01e0953231703ec116e0.jpg"},{"id":90417541,"identity":"cb783835-44cd-4f00-940b-394cb4ba760d","added_by":"auto","created_at":"2025-09-02 13:30:55","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":332100,"visible":true,"origin":"","legend":"\u003cp\u003ea. A fertilized and unfertilized ovule after \u003cem\u003ein vitro\u003c/em\u003eovular pollination\u003c/p\u003e\n\u003cp\u003eb. A \u003cem\u003ein vitro\u003c/em\u003e fertilized seed showing the characterized mottling as seen in\u003cem\u003e vivo\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003efertilized mature seeds\u003c/p\u003e\n\u003cp\u003ec. Embryo development without seed coat and endosperm formation\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/1ed3e51a38a9ab743e153581.png"},{"id":90416355,"identity":"8ffdff63-261d-4302-abae-b4c3d39f6468","added_by":"auto","created_at":"2025-09-02 13:22:55","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":396728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e. Cross section of fertilised seeds showing well developed seed coat, single celled zygote and primary\u003c/p\u003e\n\u003cp\u003eendosperm nuclei\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e. Abundance of starch grains in the nucellar area\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec\u003c/strong\u003e. Abundance of lipids in the nucellar area\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/d95edb6065b4592ce38c948c.png"},{"id":90416357,"identity":"2f106bbe-fe8d-49cf-9b28-4a7235d2e7a8","added_by":"auto","created_at":"2025-09-02 13:22:55","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":170173,"visible":true,"origin":"","legend":"\u003cp\u003ea. \u003cem\u003ein vitro \u003c/em\u003efertilised embryo after 2 months in culture\u003c/p\u003e\n\u003cp\u003eb. An \u003cem\u003ein vivo \u003c/em\u003efertilised embryo after 2 months in culture (x 100)\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/f835616f2ac486d7d4453134.png"},{"id":90416103,"identity":"b0bd072b-91de-4f9e-bed5-9e1340342380","added_by":"auto","created_at":"2025-09-02 13:14:55","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":532477,"visible":true,"origin":"","legend":"\u003cp\u003ea Stages of fruit development with corresponding ovules from 2-12 weeks\u003c/p\u003e\n\u003cp\u003eb Developmental stages of fruits (15-18 weeks old) with corresponding seeds and embryos\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/154843b293e182eb3d3f0f67.png"},{"id":90418760,"identity":"2b9742e8-a8bc-4e44-84c7-51502ed43977","added_by":"auto","created_at":"2025-09-02 13:39:05","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":533613,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTypes of endosperm development observed in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHevea\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. \u003c/strong\u003eAbsence of endosperm and embryo in mature seeds of \u003cem\u003eH.brasiliensis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eb\u0026amp; c. A fully developed embryo with partially developed endosperm\u003c/p\u003e\n\u003cp\u003ed. Development of seedling from a fruit without endosperm development\u003c/p\u003e\n\u003cp\u003ee. Profuse development of endosperm from immature fruits\u003c/p\u003e\n\u003cp\u003ef \u0026amp;g. Embryos with partially developed endosperm from immature fruit culture\u003c/p\u003e","description":"","filename":"12.png","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/332d966574cb771a77302e6d.png"},{"id":91594171,"identity":"46239b7c-f1e5-4f94-976e-fb4d18f7e8f3","added_by":"auto","created_at":"2025-09-18 07:19:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6165262,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/c3859b77-e409-46cb-a9fb-d06d3615f4da.pdf"},{"id":90416090,"identity":"83323b1e-9f85-408a-8962-6c15d0b624d3","added_by":"auto","created_at":"2025-09-02 13:14:55","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":93020,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.docx","url":"https://assets-eu.researchsquare.com/files/rs-7080763/v1/5f7d22fbdf1c03274a117565.docx"}],"financialInterests":"","formattedTitle":"Zygotic development and endosperm dynamics in Hevea brasiliensis: Progress, challenges and emerging opportunities of in vitro pollination and fertilisation","fulltext":[{"header":"Key Message","content":"\u003cp\u003e\u003cstrong\u003eThe process of Pollination, fertilization\u003c/strong\u003e\u003cstrong\u003e, Zygotic embryogenesis, endosperm development and seed formation were studied under\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eboth in vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;conditions in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eHevea brasiliensis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e. Poor endosperm development was identified as a major factor contributing to the low seed set in this species.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;pollination experiments were successful, leading to fertilization and subsequent embryo formation. Under\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;conditions, there is higher possibility of recovery of more number of hybrids, since the embryos without endosperm development can also be rescued.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe Para rubber tree (\u003cem\u003eHevea brasiliensis\u003c/em\u003e Muell. Arg.) is a commercially important species known for producing high-quality natural rubber latex, a strategic raw material with diverse industrial application (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the 7500 species of rubber yielding plants, \u003cem\u003eH. brasiliensis\u003c/em\u003e is the major contributor towards the global production of natural rubber. The demand for natural rubber always shows an escalating trend and improving the productivity through developing most suitable clones with enhanced yield coupled with the prevailing biotic and abiotic stress tolerance is the need of the hour. Tree requires at least 4\u0026ndash;5 years to attain the ripeness to blooming and retains the capacity thereafter.\u003c/p\u003e\u003cp\u003eThe recombination and selection cycles orienting towards enhanced growth and latex yield as well as the introgression of genes from wild populations to widen the genetic base through conventional crossing in rubber tree is cumbersome and laborious due to several factors like low fruit set, seasonal flowering, inaccessibility of flowers and disease infestations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHigh yielding rubber clones are being selected from breeding populations consists of mainly full-sib progenies, through lengthy conventional crossing followed by selection cycles (Simmonds, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Licy et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Priyadarshan and Clemet-Demang, 2004). Low fruit set, seasonal flowering, non-synchrony and inaccessibility of flowers and disease infestations are the major constraints in \u003cem\u003eHevea\u003c/em\u003e breeding. Low fruit set prevalent in the female parents often results in the loss of numerous potentially good cross combinations in breeding programs. Consequent reduction in the family size and recombination frequency of full-sib families results in inefficient selection, thus limiting the progress of crop improvement (Mydin et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Hand pollination program is labor intensive, and the number of crosses that can be done in a season is limited due to the inaccessibility of flowers and non-synchrony (Gireesh et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Despite the efforts to circumvent these problems, the fruit set could not be raised beyond five percent (Mydin 1989). On the other hand fixing of elite F1 genotypic selections through vegetative means could capture promising gene combination, which is considered as the major advantage of \u003cem\u003eHevea\u003c/em\u003e breeding.\u003c/p\u003e\u003cp\u003eThe presently cultivated rubber clones in Asia and Africa which contributes towards natural rubber production globally are successors of seedlings descended from Brazil to South-east Asia (Wickham collection) through Kew Garden and later undergone several cycles of breeding and selection and hence most of the present cultivars had a narrow genetic base. Reports suggests that the yield levels achievable through this currently popular Wickham gene pool is limited (James et al. 2021), warranting wide/interspecific hybridization and introgression of genes from wild populations to widen the genetic base through conventional breeding. Under such circumstances better recovery of hybrid seeds becomes inevitable and more imperative. The present study aims to examine the reasons for low fruit set and develop alternate strategies for increasing the recovery of recombinants.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eb. A new flush with immature inflorescence developed from the axils\u003c/p\u003e\u003cp\u003eFlowering in \u003cem\u003eHevea\u003c/em\u003e is seasonal following the process of defoliation and refoliation during winter. Rubber tree is monoecious with both staminate and pistillate flowers in the same inflorescence which are developed in the axils of basal leaves of newly developed shoots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003ea \u0026amp;b). Extend of flowering and seed set largely depends on the geographical location and the prevailing climatic factors. Generally flowering occurs during March-April in the northern hemisphere and September to October in the southern hemisphere (Priyadharsan 2011). In Kerala flowering follows wintering during the period from January to March. Flowering is dichogamous with incomplete protandry. Male flowers mature earlier than female flowers. The emergence of few male flowers after the maturation of female flowers makes the protandry incomplete. Female flowers are comparatively fewer and larger than male flowers and are born at the end of main branches and central axis. They are pentamerous with tricarpelary ovary (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Male flowers are numerous and smaller and seen in other parts of the panicle. The conjoined anthers often arranged in two rows over a staminal column. According to previous studies (Rao, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1961\u003c/span\u003e), \u003cem\u003eHevea\u003c/em\u003e is insect pollinated mainly by midges belonging to Ceratopogonidae and predominantly out crossed.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eimmature female flowers\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eb. Developing immature seeds separated from the ovule\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ec. T.S of \u003cem\u003eHevea\u003c/em\u003e fruit with three developing seeds\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eMature \u003cem\u003eHevea\u003c/em\u003e fruit is a three lobbed capsule with a woody endocarp, a leathery mesocarp and contains three seeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b\u0026amp;c). It will take 140 days from pollination to seed dispersal (Priyadarsan, 2011). Under open pollination, among the numerous female flowers developed, only 25% form fruits and among which only 25% will reaches maturity. In controlled pollinations pollinator ensures maximum pollination, the success rate varies from 5\u0026ndash;10 percent. In the present scenario of unprecedented climatic vagaries the availability of flowers for hand pollination itself is less. Since the peak flowering time coincides with the hot summer in Kerala which makes hand pollination difficult. The inaccessibility of flowers is another problem. Practically it is possible to do only about 70\u0026ndash;100 pollinations per person per day on the tree top. The maximum possible pollinations that can be achieved in a season are nearly 5000\u0026ndash;6000. That will account for around 250\u0026ndash;300 hybrid seeds per year. In order to carryout effective selection, the family size is important. In a heterozygous species, large family size ensures maximum combinations of alleles and the best combination can be identified and selected. Thus, it is of high importance to study the fertilization event under \u003cem\u003ein vitro\u003c/em\u003e conditions with a view to explore the possibilities of \u003cem\u003ein vitro\u003c/em\u003e fertilization and to discuss its implications for optimizing the recovery of hybrid plantlets.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe technique of \u003cem\u003ein vitro\u003c/em\u003e pollination and fertilization is a novel approach that has been used for overcoming pre-fertilization barriers (Rangaswamy and Shivanna, 1967; Valsala et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and post fertilization barriers (Sahijram, 2013). \u003cem\u003eIn vitro\u003c/em\u003e pollination and fertilization system has been accomplished in more than 57 species belonging to 14 families including Brassicacea, Caryophyllaceae, Liliaceae, Papaveraceae, Primulacea\u003cem\u003ee\u003c/em\u003e and Solanaceae (Zentkler, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1992\u003c/span\u003e and 1994). Various methods like developing interspecific and intergeneric hybrids (Zentkler, 1980; Chin et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), generation of haploids (Hess and Wagner, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Musiał and Przywara, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), and pollen tube-female gametophyte interactions and the cellular events connected to fertilization and embryo development (Dupuis and Dumas 1989; Fernando et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) have been explored. Additionally, since the IVF system allows pollen and female gametophyte to be maintained in culture, for long periods, thus genetic transformations can be carried out on sperm or eggs coupled with hybridization (Fernando et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Even though a vast literature is available on IVF systems in other plant species, \u003cem\u003ein vitro\u003c/em\u003e fertilization studies in \u003cem\u003eHevea\u003c/em\u003e is meagre. A successful IVF system is relevant in a highly heterozygous perennial crop like \u003cem\u003eHevea\u003c/em\u003e where the breeding programmes are often delayed due to the seasonal flowering, low fruit set and early fruit drop. In this context, the present study was undertaken to elucidate the dynamics of zygote and endosperm development in \u003cem\u003eHevea brasiliensis\u003c/em\u003e, and to identify potential post-fertilization barriers inherent to the species. Based on insights gained from preliminary investigations (Rekha et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) further experiments were conducted to standardize an \u003cem\u003ein vitro\u003c/em\u003e pollination and fertilization system for \u003cem\u003eHevea brasiliensis\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e1. In vitro pollination and fertilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Pollen pistil interaction studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore going for \u003cem\u003eIn vitro\u003c/em\u003e fertilization experiments, we have studied the pollen pistil interaction after pollination under in vitro conditions. The fluorescent technique reported by Kho and Baer (1968) was adopted for pollen pistil interaction studies. The pollinated flowers were fixed in FAA (Johansen, \u003cspan class=\"CitationRef\"\u003e1940\u003c/span\u003e) at different intervals 12, 24 and 48 hours after pollination. After 24 hrs, the buds were transferred to 1N NaOH for eight hours at room temperature. After thorough washing with distilled water, the buds were stained in 0.1 per cent aniline blue in 0.1 N K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e for period of 18 hrs. The pistil was later macerated in 90 percent glycerol. \u003cem\u003eIn vivo\u003c/em\u003e pollen tube growth was then examined under the fluorescent microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Standardization of pollination techniques in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAge of the flower bud\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was performed using the flowers of the Indian clone RRII 105. Female flowers at different developmental stages from one to three days before the anthesis (1DBA, 2 DBA, and 3 DBA), anthesis day (AD), and one day after anthesis (1 DAA) were used for \u003cem\u003ein vitro\u003c/em\u003e pollination. Male flowers bearing pollen grains at the late uninucleate to early binucleate stage were selected for the experiment. Flower buds were collected in the early morning and washed in running tap water for three minutes. They were then sterilized using 0.1% HgCl\u003csub\u003e2\u003c/sub\u003e for five minutes and then thoroughly washed with sterile distilled water 4\u0026ndash;5 times. The flowers were then blotted on a sterile filter paper to remove the moisture. Anthers were dissected from surface sterilized male flowers using fine forceps.The flowers were then blotted on a sterile filter paper to remove the moisture. Anthers were dissected from surface sterilized male flowers using fine forceps.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC. Method of pollination\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e self-pollination was done using different techniques described by Bhojwani and Razdan (\u003cspan class=\"CitationRef\"\u003e1983\u003c/span\u003e) \u003cem\u003eviz\u003c/em\u003e., (i) \u003cem\u003eIn vitro\u003c/em\u003e stigmatic pollination: the female flowers after sterilization were inoculated as such in the nutrient medium. Anthers dissected from the male flowers were placed on the stigma of the female flower; (ii) Intra ovarian pollination: After surface sterilization, the perianth was completely removed, and the stigma was excised to expose the ovary. Pollination was carried out directly on the cut surface of the ovary. (iii) \u003cem\u003eIn vitro\u003c/em\u003e placental pollination: Following the careful removal of the perianth, stigma, and ovary wall, the placenta bearing the ovules was exposed. Pollen was then applied directly onto the ovules under sterile conditions. (iv) \u003cem\u003eIn vitro\u003c/em\u003e ovular pollination/test tube fertilization: The perianth, stigma and the ovary wall were removed carefully and the ovule supported by the funicle were isolated and inoculated in the medium. The anthers dissected from male flowers were placed close to the ovules. Test tube fertilization was performed as described below in four different ways. \u003cem\u003eMethod 1\u003c/em\u003e: The ovules were inoculated in the medium and the anthers were kept aside. \u003cem\u003eMethod 2\u003c/em\u003e: Ovules were inoculated in the medium and anthers were kept aside and few drops of sucrose solution were poured over them. \u003cem\u003eMethod 3\u003c/em\u003e: Ovules were inoculated in the medium and pollinated with anthers and then few drops of pollen germination medium were applied over them. \u003cem\u003eMethod 4\u003c/em\u003e: The ovules were immersed in 1% CaCl\u003csub\u003e2\u003c/sub\u003e for one day and pollinated with freshly isolated anthers. The pollinated flowers/ovaries/ovules were cultured on media in Petri dishes and sealed with Para film and incubated under dark conditions at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003eo\u003c/sup\u003eC. Unpollinated flowers/ovaries/ovules were also inoculated in the same culture medium as control. After one week, the swelled ovaries were counted as fertilized and they were transferred to new culture media in Petri dishes for embryo development and sealed with Para film and incubated under dark condition at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003eo\u003c/sup\u003eC. Observations were recorded at weekly intervals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Culture media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe basal salts of MS, WPM (Lloyd, \u003cspan class=\"CitationRef\"\u003e1980\u003c/span\u003e) and N6 (Nitsch and Nitsch, \u003cspan class=\"CitationRef\"\u003e1969\u003c/span\u003e) were experimented initially, for the growth of fertilized ovules. Based on the swelling of ovules, the basal medium was fixed and varying concentrations of sucrose from 3\u0026ndash;18% were experimented. After fixing the sucrose level, combinations of growth regulators such as BA, KIN, GA\u003csub\u003e3\u003c/sub\u003e, NAA and 2, 4-D were tried for the growth of fertilized ovules.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3. Histological studies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ovules showing morphological similarities with \u003cem\u003ein vivo\u003c/em\u003e seeds were recovered and fixed in FAA and processed for microtome sectioning. For general histology, sections were stained with Periodic Acid Schiff\u0026rsquo;s reagent (Jensen, \u003cspan class=\"CitationRef\"\u003e1962\u003c/span\u003e). For detecting starch, I\u003csub\u003e2\u003c/sub\u003eKI treatment and to localize lipids, Sudan IV dye (Johansen, \u003cspan class=\"CitationRef\"\u003e1940\u003c/span\u003e) was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4. Comparison of\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003ein vivo\u003c/strong\u003e \u003cstrong\u003efertilised ovules\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ea. Performance of in vitro and in vivo fertilised ovules in culture\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn order to compare the growth of \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e fertilised ovules under \u003cem\u003ein vitro\u003c/em\u003e culture conditions, \u003cem\u003ein vivo\u003c/em\u003e pollination was performed as per standard procedure (Mydin and Saraswathiyamma, 2005) and the pollinated flowers were collected after three days and sterilized as described in the session 3.b. Ovules were isolated from these flowers and inoculated in the same medium and growth was observed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eb. Growth of the fruit, endosperm and embryo under field conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFruits at different maturity were collected from the trees and, the developing ovules were dissected and observed for embryo/endosperm development, in order to study the endosperm growth under field conditions. The developing seeds were collected from fruits of different maturity and cultured in nutrient medium and observed for the development of embryo and endosperm.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ec.\u003c/em\u003e Growth and development of immature seeds collected from the field grown trees and cultured \u003cem\u003ein vitro\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eOpen pollinated fruits from the clone RRII 105 at different stages of maturity were collected and inoculated in media following \u003cem\u003ehalf ovulo\u003c/em\u003e embryo culture (Rekha et al. \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003e1. In vitro pollination and fertilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea. Pollen-Pistil interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSuccess of pollination depends on the sequential events starting from the acceptance of pollen by the stigma and a complex and cooperative interaction between viable pollen and a receptive pistil followed by pollen tube growth through the pistil to the ovule. The pollen pistil interaction studies indicated that there is profuse development of pollen tube after pollination and it enters the ovules 24\u0026ndash;48 hours after pollination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e) in all the samples tested. These results are in conformity with earlier reports by Sedgley and Attanayake (Sedgly and Attanayake, 1986). In flowering plants, the transport of non-motile sperm to distant egg cells is required for effecting double fertilization. The pollen tube penetration of the pistil and maintenance of structural integrity of the pollen tube while moving to the destination following the positional guidance cues is required for successful fertilization event. During the penetration of pistil, the pollen tube encounters numerous physical barriers preventing the ultimate fusion between male and female gametes. Studies on pollen- pistil interactions serve as a distinctive model for elucidating the mechanisms of plant cell to cell interaction and signaling, which are critical for successful fertilization. In the present study, the sufficient growth of pollen tube ensures the absence of any pre-fertilization inhibitors in the stigma/style leading to absence of seed set.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Age of the flower buds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmong the five developmental stages assessed for in vitro pollination, the highest percentage of ovule swelling, an indicator of successful pollination, was recorded in female flowers collected one day before anthesis (1 DBA) at 79.2%, followed closely by those collected on the day of anthesis (AD) with 75.2% success (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea \u0026amp; b). Flowers collected one day after anthesis (1 DAA) showed a moderate success rate of 58%. In contrast, significantly lower percentages were observed in flowers collected 2 and 3 days before anthesis, indicating these stages were less responsive to in vitro pollination.This indicates that the stage of the flower is an important factor in determining the success of fertilization and it is highly specific. Immature flowers are not suitable for pollination. Fertilization success is largely determined by stigma receptivity, with the timing and duration of receptivity playing a critical role in effective pollination and subsequent seed set. It may be assumed that lack of stigma receptivity may be the reason for low fertilization success in immature flowers. In contrast, higher number of seeds was produced in \u003cem\u003eLilium\u003c/em\u003e when the flowers three days prior to anthesis were pollinated (Van Tuyl et al., \u003cspan class=\"CitationRef\"\u003e1991\u003c/span\u003e). Stigma receptivity is a species character and it varies among different species of same genus. In Eucalyptus, the stigma receptivity varies from 5 to 10 days among different species (Pauldasan et al \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Flowers on the day of anthesis or one or two days after anthesis improved seed set in crops like Ginger, Papaver, Nicotiana and Maize (Valsala et al. \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e; Kanta and Mahaswari 1963; Rangaswamy et al. 1967; Gengenbach, \u003cspan class=\"CitationRef\"\u003e1977\u003c/span\u003e). In legumes like \u003cem\u003eSwainsona formosa\u003c/em\u003e also stigma receptivity started one day before anthesis (Zulkarnain et al. \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Even though no significant difference was noticed in the success rate of the male flowers on one day before anthesis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ea\u0026amp;b) and on the day of anthesis in \u003cem\u003eH. brasiliensis\u003c/em\u003e, the rate of contamination was high for the flowers taken on the day of anthesis. Similarly, a high fertilization success (58%) was noticed in female flowers one day after anthesis when compared to 2 DBA and 3 DBA but the rate of contamination was high. Based on these observations, the flowers one day prior to anthesis were utilized for further experiments.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffect of flower bud age on fertilization success after \u003cem\u003ein vitro\u003c/em\u003e pollination in \u003cem\u003eHevea brasiliensis\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eTreatments\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAge of the flower bud\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSuccess (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 DBA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 DBA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e46.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1 DBA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e79.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAnthesis day\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e75.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eOne DAA\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e58.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eCD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.2\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003eDBA: days before anthesis; DAA: days after anthesis; values\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"3\"\u003eare means of multiple observations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003ec. Methods of pollination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of different methods of pollination on fertilization success as indicated by ovary/ovule/ development are presented in the Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Based on the observations two weeks after pollination, the following results were derived. Among the four methods of pollination tried, \u003cem\u003ein vitro\u003c/em\u003e stigmatic pollination and test tube fertilization showed some positive results, as indicated by the swelling of ovaries and ovules respectively after fertilization. The intra ovarian pollination and \u003cem\u003ein vitro\u003c/em\u003e placental pollination failed to give any positive results and the ovules dried within a few days. Even though 40% of ovaries were enlarged under \u003cem\u003ein vitro\u003c/em\u003e stigmatic pollination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), none of them contained seeds. In \u003cem\u003eHevea\u003c/em\u003e, the studies on pollen pistil interaction indicated that the pollen tubes enter the ovules 24\u0026ndash;48 hours after pollination. This ensures the absence of any pre-fertilization inhibitions, for seed set. The fertilization may not have taken place due to some incompatibility or the embryo might have aborted at an early stage due to some post fertilization inhibitions. This observation is comparable to the available reports under \u003cem\u003ein vivo\u003c/em\u003e conditions (Sedgley and Attanayake, \u003cspan class=\"CitationRef\"\u003e1986\u003c/span\u003e). In \u003cem\u003eHevea\u003c/em\u003e, there are reports that the control mechanisms for fruit wall formation and embryo growth are different and independent (Priyadharsan 2011). Hence it is possible that fruit wall may grow even after embryo abortion. Lack of some essential nutritional factors can also be attributed to the absence of seeds in \u003cem\u003ein vitro\u003c/em\u003e stigmatic pollination of \u003cem\u003eHevea\u003c/em\u003e. Development of fruits after stigmatic, stylar and intra ovarian pollination without seeds is reported in the \u003cem\u003ein vitro\u003c/em\u003e fertilization of ginger (Valsalaet al. \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e). In contrast, the application of pistil culture and in vitro stigmatic pollination techniques significantly improved seed set in \u003cem\u003ePetunia\u003c/em\u003e and (Rangaswamy et al. 1967; Usha, \u003cspan class=\"CitationRef\"\u003e1965\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eEffect of pollination method on \u003cem\u003ein vitro\u003c/em\u003e pollination of \u003cem\u003eHevea brasiliensis\u003c/em\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMethod of pollination\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCultures with ovary\u003c/p\u003e\n\u003cp\u003edevelopment (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCultures with ovule\u003c/p\u003e\n\u003cp\u003edevelopment (%)\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStigmatic\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e placental\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIntra-ovarian\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.0\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTest tube\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e--\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e80\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the different methods tried for test tube fertilisation, immersing the ovules in 1% CaCl2 for 24 hrs followed by pollination and pouring pollen germination solution over the pollinated ovules was promising. 80% of the pollinated ovules swelled. Inoculating the ovules in the medium and keeping anthers over them didn\u0026rsquo;t work. Pouring sucrose solution after pollination resulted in the swelling of 10\u0026ndash;12 percent ovules. However these ovules dried after a few days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed. Growth and development of ovules after test tube fertilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the current study, after \u003cem\u003ein vitro\u003c/em\u003e ovular pollination (test tube fertilization), about 80% of the ovules enlarged two weeks after pollination. The fertilized ovules were ivory colored and opaque, whereas the unfertilized ovules were glassy in appearance, turned brown and dried after a few days (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea). The unpollinated controls also turned brown within a few days. The swelled ovules were further cultured in different media combinations. Among the swelled ovules, further development was noticed in 40% of ovules. The rest of the ovules dried off after one month. Among the remaining swelled ovules, two types of developments were noticed wherein the first 10% ovules as a whole developed into small seeds. At 30 days post-fertilization, the in vitro-derived ovule attained a size similar to its in vivo counterpart, although its morphological characteristics closely resembled those of a mature seed. After 30 days, the size of the \u003cem\u003ein vitro\u003c/em\u003e raised fertilized ovule was comparable with \u003cem\u003ein vivo\u003c/em\u003e ovule but the morphology of the \u003cem\u003ein vitro\u003c/em\u003e developing ovule resembled a mature seed under field conditions. The seed coat is sclerified and characteristic mottling was observed in the surface of these ovules as in the case of mature seeds (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e.b). Since further embryo growth could not be observed in these cultures, they were fixed for anatomical studies.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe remaining 30% of the ovules showed a different pattern of development. The embryos were developed without endosperm and seed coat formation (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec). By approximately 30 days after pollination, the integuments were observed to be displaced, exposing embryos at the globular stage. This is in contrast to the development of seeds under \u003cem\u003ein vivo\u003c/em\u003e conditions. The lack of endosperm formation or its early degradation during the zygote or young embryo stage has been documented in several plant species, often posing a major constraint to successful embryo development (Kapoor, \u003cspan class=\"CitationRef\"\u003e1959\u003c/span\u003e; T\u0026ouml;pfer and Steinbiss, \u003cspan class=\"CitationRef\"\u003e1985\u003c/span\u003e; Zenkteler and Nitsche, 1985). In the interspecific crosses of brassicas also absence of endosperm is observed after \u003cem\u003ein vitro\u003c/em\u003e pollination. However, these embryos could be developed in to plantlets.\u003c/p\u003e\n\u003cp\u003eThe absence of endosperm may be either due to the failure of triple fusion during fertilization or due to endosperm abortion due to embryo endosperm incompatibility. Otherwise, after fusion, triploid nuclei may be degenerated due to some unknown reasons. The lack of proper nutrition can also contribute to the absence of endosperm. Another possible reason may be the suppression of the triploid endosperm by the faster growth of the diploid embryo. The development of embryo and germination of seedlings from the embryos without endosperm formation after IVF has been reported in Chicory (Castano, 2000). Similarly, in the test tube fertilization of \u003cem\u003eEschscholzia californica\u003c/em\u003e the embryo showed normal development during its early growth whereas the endosperm was purely developed and degenerated. This indicates the possibility of recovery of seedlings from such embryos in \u003cem\u003eHevea\u003c/em\u003e also. Optimization of nutritional requirements will be worth trying for saving these embryos. A few embryos could be recovered from these cultures and they were grown up to the cotyledonary stage. However, the size of the embryos was very small.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee. Histological observations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fertilized ovules, with seed coat development were subjected to anatomical studies in order to understand the development pattern. The anatomical studies of the ovules 90 days after pollination revealed the presence of two layered outer walls (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e.a). Well-developed seed coat with a thin walled inner zone with radially elongated sclerified cells could be seen at this stage. Seed coat is single layered in the chalazal region. The abundance of starch and lipids were observed in the nucellar area (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e.b. \u0026amp; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003ec). The zygote and primary endosperm nuclei at single cell stage were also visible. This indicates successful fertilization after \u003cem\u003ein vitro\u003c/em\u003e pollination. When an ovule of the same age after \u003cem\u003ein vivo\u003c/em\u003e pollination was observed, the seed coat was soft and ivory in color and not similar to \u003cem\u003ein vitro\u003c/em\u003e in the case of seed coat. Under \u003cem\u003ein vitro\u003c/em\u003e conditions, the seed coat formation seems to be faster and has been almost completed in about 90 days. The hard seed coat may be preventing the absorption of nutrients from the medium which may in turn effect further development of the embryo. Subsequent experiments will focus on the removal of integuments at defined intervals following pollination.\u003c/p\u003e\n\u003cp\u003eThe pollination success as indicated by the swelling of ovules under both \u003cem\u003ein vivo/ in vitro\u003c/em\u003e conditions is presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. In the \u003cem\u003ein vivo\u003c/em\u003e pollination followed by \u003cem\u003ein vitro\u003c/em\u003e culture, an initial success of 67.5% based on the swelling of ovules was achieved after 2 weeks of culture. Among these, only 19 percent of swollen ovules showed continued growth and rest of them dried off. In the case of \u003cem\u003ein vitro\u003c/em\u003e fertilised ovules, among the 80% of swelled ovules 41 percent showed sustained growth after 1 month. Subsequently, they showed two types of growth pattern as described earlier. A few embryos in both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e conditions developed up to cotyledonary stage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). Even though the initial success is comparable, more ovules showed sustained growth after IVF, compared to \u003cem\u003ein vivo\u003c/em\u003e fertilization. After initial swelling of the ovules, the possibility of early embryo abortion cannot be ruled out in this situation. The sudden stress induced due to the detachment of pollinated flowers from the tree and the sterilization process might have triggered early embryo abortion. The in vitro-fertilized embryos were smaller in size compared to the in vivo-fertilized embryos.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePollination success under \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e pollination as indicated by the swelling of ovules\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eMethods of pollination\u003c/p\u003e\n\u003c/th\u003e\n\u003cth colspan=\"2\" align=\"left\"\u003e\n\u003cp\u003ePercentage of ovules swelled\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAfter 14 days\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAfter 30 days\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e pollination\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e19\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTest tube fertilization\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e79.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eT stat\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e34.79\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003ee. Culture medium\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe success of \u003cem\u003ein vitro\u003c/em\u003e pollination depends on the formulation of appropriate medium, which will support pollen germination and tube growth leading to fertilization and zygote development. MS basal salts supplemented with 100 mg L\u003csup\u003e-1\u003c/sup\u003e boric acid and 20 gL\u003csup\u003e-1\u003c/sup\u003e sucrose promoted pollen germination and effective fertilization, as indicated by swelling of ovules. Among various basal media and concentrations of sucrose tried out, more number of swelled ovules were obtained in MS medium supplemented with 50 gL\u003csup\u003e-1\u003c/sup\u003esucrose. The influence of sucrose on embryo development is well studied in other crops. In \u003cem\u003ePrunus\u003c/em\u003e, \u003cem\u003ein vitro\u003c/em\u003e pollination followed by culture on a medium containing 15% sucrose resulted in a high frequency of embryo development. (Dziedzic et al. \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e). In the media, for ovule growth, organic supplements \u003cem\u003eviz.\u003c/em\u003e, CW (20%), malt extract (200 mg/l), casein hydrolysate (400 mg/l) and banana powder (100 mg/l) was added as it was found to be good for the rescue of embryos from immature fruits in \u003cem\u003eHevea\u003c/em\u003e (Rekha et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Among the five growth regulators tested, Kinetin (2mgL\u003csup\u003e-1)\u003c/sup\u003e and GA\u003csub\u003e3 (\u003c/sub\u003e2mgL\u003csup\u003e-1\u003c/sup\u003e) were found to exhibit positive results when supplied individually. Other growth regulators such as 2, 4-D, NAA and BA supplemented in the medium, resulted in callus induction from the outer integument and prevented further development of the ovule. However, when kinetin (2.0 mg L\u003csup\u003e-1\u003c/sup\u003e) was combined with 2mgL\u003csup\u003e-1\u003c/sup\u003e GA3, the growth of the fertilized ovule was improved. A few embryos could be grown up to cotyledonary stage (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). Further increase in concentrations of both had an adverse effect on ovule development. Addition of Kinetin promoted ovule development after \u003cem\u003ein vitro\u003c/em\u003e pollination of two different species in Brassicas (Sosnowska and Teresa Cegielska-Taras, 2014). Kinetin had a positive effect on the growth of immature embryos rescued from field in \u003cem\u003eH. brasiliensis.\u003c/em\u003e Experiments are underway to improve the nutritional status of the medium, for achieving further growth of the embryo. Standardization of the media requirements is very difficult using IVF embryos since tangible number of embryos could not be recovered with the present success rate. Hence experiments are being performed using open pollinated fruits. In \u003cem\u003eHevea\u003c/em\u003e, embryo culture was successful from 8 week old fruits onwards. Experiments are underway for the media optimization and culture requirements for very immature fruits (1\u0026ndash;8 weeks old). Once this is achieved, the same can be used for IVF embryos as well.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ef. Endosperm development under\u003c/strong\u003e \u003cstrong\u003ein vitro\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003ein vivo\u003c/strong\u003e \u003cstrong\u003econditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhen different stages of fruits (1\u0026ndash;5 months) were dissected, it was observed that the embryos are visible only in later stages of fruit development (4\u0026ndash;5 months). The different stages of fruit development (2\u0026ndash;12 weeks) with their corresponding seeds are given in the Fig.\u0026nbsp;11. It is observed that, in \u003cem\u003eHevea\u003c/em\u003e, the growth of the fruits and seeds follow a specific pattern. The fruit wall grows faster and by 2\u0026ndash;3 months it becomes thickened. After thickening of the fruit wall, the seeds will starts developing and when it fills the locules, the endosperm development initiates. After the completion of endosperm development, the embryo starts developing. By this time, the fruits will attain maximum growth, seed coat will be thickened and the endosperm will be fully developed. Once the development is initiated, the embryos grow faster and a fully developed embryo with cotyledons intruding in to the endosperm will be formed within 10\u0026ndash;15 days. Different stages of fruits 2\u0026ndash;10 weeks old) with their corresponding ovules are given in Fig.\u0026nbsp;11a. No embryos are visible in any of these stages. Fruits of 15\u0026ndash;18 weeks maturity are shown in Fig.\u0026nbsp;11b. In \u003cem\u003eH.brasiliensis\u003c/em\u003e, it is reported that, under field conditions, the embryos will be small and microscopic till 14 weeks after fertilization (Muzick, 1954).Our results are in conformity with this. Only during the last few weeks of fruit maturity we could see the embryos (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eWhen mature seeds developed \u003cem\u003ein vivo\u003c/em\u003e are observed, many seeds are without endosperm development (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003ea). In this case, embryos also seemed to be degenerated. Partially developed endosperm was also observed in some seeds where the embryos are live and fully developed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003eb \u0026amp;c).In immature fruit culture, it was observed that endosperm development is partial or completely absent in many of the ovules. However, embryos will grow normally in culture even in the absence of endosperm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e.d) and plantlets could be recovered from these embryos. These observations reveal that embryo- endosperm communication is apparent under field conditions and may be attributed to the low seed set. After double fertilization, seed development initiates with the endosperm formation from the primary endosperm nucleus and development of embryo from the zygote. It is assumed that the development of endosperm and embryo is happening in coordination under \u003cem\u003ein vivo\u003c/em\u003e condition. Although embryo and endosperm development are distinct processes, proper seed formation depends on elaborate and synchronized communication between the two. There are accumulating evidences which demonstrates the requirement of certain protein regulators originating from the endosperm for proper development of the embryo (Ingram et al. 2020). The ratio of maternal to paternal gene products in the endosperm plays a critical role in determining the success of crosses between individuals of differing ploidy levels within a species, as well as interspecific crosses. Maternally expressed small-interfering RNAs (siRNAs) are believed to regulate this balance by targeting growth-promoting genes, thereby influencing endosperm development and hybrid viability (David Haig, \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). The endosperm acts as a major metabolic sink for absorbing nutrients from the maternal tissues and sequestering them in the central vacuole. Nutrients are then re-exported to the embryo from the endosperm (Ingram et al. 2020). In \u003cem\u003eHevea\u003c/em\u003e, early fruit drop is common under natural conditions as well as after hand pollination. These observations reveal that embryo-endosperm communication is apparent under field conditions and may be attributed to the low seed set. In the present experiment, after in vitro fertilization a few embryos developed without endosperm development. When mature fruits from the field are observed, there also endosperm is absent/partially developed. These observations lead to the assumption that failure of endosperm may be one of the main reasons for low fruit set. Hence future investigations focusing on these aspects need to be carried out. In immature fruit culture, the embryos developed with /without endosperm but plant regeneration was successful from both cases. Embryo rescue and culture is most successful in the recovery of plants where fruit drop is caused due to endosperm degradation. It is generally presumed that the tissue culture medium compensates for the absence of endosperm by supplying the essential nutrients required for embryo development.Continued refinements in culture media have permitted younger and smaller embryos to be rescued and grown to maturity in many crops (Stewart, \u003cspan class=\"CitationRef\"\u003e1981\u003c/span\u003e). In our experiments also, embryos could be rescued and successfully regenerated in to plantlets.\u003c/p\u003e\n\u003cp\u003eUnder \u003cem\u003ein vitr\u003c/em\u003eo conditions, the embryo/endosperm dependency is not visible and many of the embryos develop into a seedling surpassing the endosperm influence. This opens up the possibility of recovering more number of hybrid seedlings by \u003cem\u003ein vitro\u003c/em\u003e pollination. In \u003cem\u003eHevea\u003c/em\u003e, early fruit drop is a common phenomenon under natural conditions as well as during artificial pollination. Comparable embryo development was observed when fertilized ovules derived from in vivo pollination were subsequently cultured under identical conditions. In Spite of high initial success (67%), the value was substantially reduced (17%) (Table.3).This may be attributed to Lack of proper communication between endosperm and embryo leading to the degeneration of either of them. However, further research is needed to confirm the hypothesis. Deriving accurate nutrient composition for promoting the independent growth of the zygote is a major challenge in the success of \u003cem\u003ein vitro\u003c/em\u003e fertilization and plant recovery.\u003c/p\u003e\n\u003cp\u003eThere is a possibility of inducing calli/secondary embryos from the zygotic embryo and uniform seedlings can be generated. Induction of zygotic polyembryoni and development of uniform seedlings are already reported in \u003cem\u003eHevea\u003c/em\u003e, using open pollinated immature seeds (Rekha et al.2015). This possibility can be applied to IVF embryos as well and multiple seedlings of desirable crosses can be generated. They enables replicated evaluation in the nursery stage itself and selection will be more effective. This is especially important in the case of perennial tree crop like \u003cem\u003eHevea\u003c/em\u003e, where long term field trials are inevitable for a meaningful selection of elite individuals. Callus induction and subsequent embryogenesis enables development of elite, uniform rootstocks. Embryogenic calli derived from the zygote is an ideal explants for gene transfer experiments, since the regeneration and hardening will be more efficient. There is also a possibility of developing triploids of desired combinations. In a triploid, 2 sets of maternal chromosomes and one set of paternal chromosomes will be there. Only for compatible crosses, endosperm develops \u003cem\u003ein vitro\u003c/em\u003e. Hence there is always a possibility of regenerating viable triploid plants through somatic embryogenesis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe present study explores the feasibility of achieving \u003cem\u003ein vitro\u003c/em\u003e pollination and fertilization in \u003cem\u003eHevea brasiliensis\u003c/em\u003e, followed by normal embryo development under controlled conditions. The results confirm successful fertilization and embryo formation, with ovules cultured up to 90 days post-fertilization, leading to embryo development up to the cotyledonary stage. Further refinements in pollination techniques, along with optimization of culture media composition and environmental conditions, are likely to enhance embryo maturation and facilitate subsequent seedling development. This approach could significantly improve the recovery rate of hybrid seedlings, thereby increasing the efficiency of selection for elite traits in breeding programs. The study also highlights that defective endosperm development or impaired endosperm-embryo communication may contribute to low fruit set under natural field conditions a limitation that is circumvented under in vitro conditions, allowing for higher plant recovery. Beyond enhancing hybrid production, a successful in vitro fertilization system offers a platform for advanced crop improvement strategies, including gene transfer, gene editing, and the development of uniform elite rootstocks.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRRII\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRubber Research Institute of India\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e fertilization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDBA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDays Before Anthesis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDays After Anthesis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAnthesis Day\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFormalin Acetic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eMS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eMurashigae and Skoog\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eWPM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eWoody Plant Medium\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSiRNAs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSmall-interfering RNAs\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBenzyl adenine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGibberellic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eNAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eNaphthalene Acetic Acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e2, 4, D\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2, 4, Dichlorophenoxy acetic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFAA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFormaline Acetic Acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCW\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCoconut Water\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCasein Hydrolysate\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe express our sincere thanks to Dr. James Jacob, former Director of Research of the Rubber Research Institute of India for the encouragement of this study. This work was supported by the Rubber Board of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the authors contributed equally to the work. \u003cem\u003eThe study was conceptualised and designed by the senior authors Rekha K and Kumari Jayasree.\u003c/em\u003e\u003cem\u003e\u0026nbsp;Material preparation\u003c/em\u003e\u003cem\u003e\u0026nbsp;was done by Raheena KT and Athira.P.V\u003c/em\u003e\u003cem\u003e, data collection and analysis were performed by\u0026nbsp;\u003c/em\u003e\u003cem\u003eVinoth Thomas, GireeshT ,Jithu George and Alex Raju\u003c/em\u003e\u003cem\u003e. The first draft of the manuscript was written by\u0026nbsp;\u003c/em\u003e\u003cem\u003eRekha K\u003c/em\u003e\u003cem\u003e\u0026nbsp;and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo additional data is associated with this manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBhojwani SS, Razdan MK (1983) Plant Tissue Culture: Theory and Practice. Elsevier Science Publishing, Amsterdam, p 194\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCasta\u0026ntilde;o CI, De Proft MP (2000) \u003cem\u003eIn vitro\u003c/em\u003e pollination of isolated ovules of \u003cem\u003eCichorium intybus\u003c/em\u003e L. 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Environ Exp Bot 21(3\u0026ndash;4):301\u0026ndash;315\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"in vitro fertilization, Hevea brasiliensis, Endosperm development, Zygotic embryogenesis, Embryo rescue, in vitro breeding","lastPublishedDoi":"10.21203/rs.3.rs-7080763/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7080763/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eHevea brasiliensis\u003c/em\u003e is the primary source of natural rubber, a vital raw material with widespread applications across various industries. Owing to the consistent rise in demand for this strategic commodity, enhancing productivity remains as a key objective in rubber breeding. However, genetic recombination breeding in \u003cem\u003eHevea\u003c/em\u003efaces significant challenges, including seasonal flowering, low fertility, early fruit drop etc. These factors limit the production of sufficient full-sib families available for effective selection and thereby increase the cost and effort of artificial pollinations. Enhancing fruit/seed set is particularly important in a highly heterozygous perennial crop like \u003cem\u003eHevea\u003c/em\u003e. This study was aimed to investigate the fertilization and fruit set processes in \u003cem\u003eHevea brasiliensis\u003c/em\u003e, identify various factors contributing to low fruit set and explore the feasibility of an \u003cem\u003ein vitro\u003c/em\u003e fertilization system to generate a higher number of desirable recombinants. The research was conducted using flowers and fruits from mature trees of clone RRII 105. Key factors such as age of the flower, methods of pollination, and culture media for fertilization and embryo formation were examined. Successful fertilization and embryo formation under \u003cem\u003ein vitro\u003c/em\u003econditions were demonstrated in \u003cem\u003eH.brasiliensis . \u003c/em\u003efor the first time. Additionally, the study provides novel insights into embryo and endosperm development under both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e conditions, shedding light on potential causes of low fruit set. The findings will be beneficial for breeders, in the recovery of hybrid seeds especially from difficult crosses of breeding programs. The protocol developed in this study can be utilized for the consistent and sustainable development of hybrid embryos into seedlings from different cross combinations.\u003c/p\u003e","manuscriptTitle":"Zygotic development and endosperm dynamics in Hevea brasiliensis: Progress, challenges and emerging opportunities of in vitro pollination and fertilisation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-02 13:14:50","doi":"10.21203/rs.3.rs-7080763/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1876b2d5-9e8e-4365-93e5-a879f4288c53","owner":[],"postedDate":"September 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-18T07:11:48+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-02 13:14:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7080763","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7080763","identity":"rs-7080763","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}