A repeatable and efficient plant regeneration protocol is crucial for applying molecular breeding methods to overcome the difficulties of conventional breeding programs to improve eggplant germplasm, such as genetic transformation and genome editing procedures (Khatun et al., 2022). Therefore, this study was conducted to develop a proficient plant regeneration method using cotyledon, hypocotyl, and leaf in two eggplant cultivars (Pusa purple long-PPL and Pusa green round-PRG). This investigation also evaluates the influence of a wide variety of factors on regeneration efficiency. The diverse factors like explants, genotypes, and manipulation of suitable concentrations and combination of the PGRs significantly affect the induction and enhancement of shoots and plant regeneration in eggplant (Saini and Kaushik, 2019; Sharma et al., 2020; Alam and Samiullah, 2021; Yesmin et al., 2021; Khatun et al., 2022). The different cytokinins like BAP, KIN, 2iP, TDZ, and ZEA were successfully employed for shoot induction and proliferation from various types of explants in diverse genotypes/cultivars of eggplant (Saini and Kaushik, 2019; Sharma et al., 2020; Alam and Samiullah, 2021; Yesmin et al., 2021; Khatun et al., 2022).
Effect of cytokinins on shoot induction
The three explant types, cotyledon, hypocotyl, and leaf, were obtained from different genotypes, Pusa purple long (PPL) and Pusa green red (PGR) of eggplant. These explants were placed on a hormone-free medium, and no response was observed except for the inducing callus formation at the cut ends. All three explants were cultured on the medium augmented with a variety of cytokinin concentrations alone. The explants exhibited signs of the initiation of adventitious shoot buds while culturing on the medium containing all concentrations of cytokinins. Cytokinins involve several physiological functions, including the formation of the shoot and/or root meristem (Hurny et al., 2020; Hnatuszko-Konka et al., 2021). The formation of adventitious buds was recorded within one to two weeks of culture. The shoot bud induction significantly relies on many factors, such as explant, type of cytokinin, and cultivar/genotype. All three explants showed the occurrence of adventitious buds within a week on the culture medium fortified with either mT or BAP. Meanwhile, the ZEA-containing medium exhibited a delayed response. Among the two cultivars examined, the PPL cultivar was significantly more responsive than the PGR. Our findings are in agreement with previous investigations on eggplant, where different regeneration responses have been observed in various genotypes of eggplant (Pawar et al., 2013; Yesmin et al., 2018; 2021; Khatun et al., 2022).
The media containing various concentrations of mT induced the maximum number of adventitious shoots and showed optimal regeneration frequency in all explants of two eggplant cultivars (Tables 1–5). The medium fortified with 11.10 µM BAP generated the optimum number of shoots in all three explants and showed the maximum regeneration response in both eggplant genotypes. The concentration of up to 11.10 µM of BAP declined the shoot number and percentage of regeneration in two genotypes of eggplant. Among the three cytokinins evaluated, the ZEA was observed as the least reactive cytokinin compared to mT and BAP in all explants of the eggplant cultivars. The shoot regeneration on medium augmented with 9.12 µM ZEA was observed as an appropriate concentration for inducing the maximum adventitious shoot buds and regeneration efficiency in the three explants of two cultivars of eggplant (Tables 1–3).
The medium with mT at 8.28 µM was a highly proficient cytokinin than BAP and ZEA, generating from 2.3 to 18.6 shoots depending on the explant type in PPL and PGR after six weeks of cultures (Table 1–3). The medium augmented with 11.10 µM BAP induced the maximum number of adventitious buds in all three explant types and showed the highest regeneration response in both genotypes of eggplant (Tables 1–3). The initiation of multiple shoots and shoot regeneration were increased gradually. The cotyledons generated 18.6 and 13.2 shoots per explant on the medium amended with mT (8.28 µM) after six weeks of culture in PPL (Fig. 1a) and PGR, respectively (Tables 1–3). In contrast, hypocotyl explants produced an average of 8.3 and 6.4 shoots/explant on the medium augmented with mT at 8.27 µM in PPL (Fig. 1b) and PGR, respectively. The leaf explants produced with adventitious shoots were recorded at 12.7 and 11.3 shoots per explant (Fig. 1c). The medium containing diverse mT concentrations enhanced the adventitious shoot buds per explant in both genotypes of eggplant compared with the other two cytokinins, BAP and ZEA, in all the explants of both cultivars of eggplant (Tables 1–3). The medium was amended with BAP at 11.10 µM, which generated the optimum adventitious shoots per explants, such as 13.4 shoots per cotyledon, 8.2 shoots per hypocotyl, and 10.6 shoots per leaf explants of the PPL cultivar. In contrast, the PGR cultivar recorded 10.9 shoots per cotyledon, 8.7 shoots per leaf explant, and 6.8 shoots per hypocotyl.
All concentrations of ZEA were found to be less significant than mT and BAP in the initiation of shoots from explant and regeneration from all explants of two cultivars of eggplant. Among various concentrations of ZEA, the medium containing 9.12 µM ZEA was shown as an efficient concentration in producing the maximum shoots per explant, and it showed the highest regeneration frequency (Tables 1–3). The cotyledons generated 9.5 and 8.7 shoots in PPL and PGR genotypes, respectively. In comparison, 7.5 shoots and 5.8 shoots in hypocotyl and 8.2 shoots and 6.2 shoots in leaf were recorded in PPL and PGR cultivars, respectively. When the cytokinin concentrations were increased, they negatively affected regeneration efficiency and reduced the number of shoots (Tables 1–3). The cotyledon explants are better responsive in initiating the number of adventitious shoots and regeneration frequency, followed by leaf explants in both genotypes. When cultured on three different cytokinins, the hypocotyl explants are least responsive in both genotypes. The mT was recorded as highly efficient as BAP and ZEA regarding the number of shoots per explant and regeneration frequency for the three explants of two eggplant genotypes.
In this study, we have compared three different cytokinins, BAP, mT, and ZEA, for their regeneration efficiency and induction of adventitious shoots in two genotypes using three explant types, cotyledon, hypocotyl, and leaf (Tables 1–3). The medium fortified with mT demonstrated the highest regeneration response and induced the optimum number of adventitious shoots. These observations are in agreement with previously reported studies on the effectiveness of the mT on the initiation of multiple shoots in many plant species like Cannabis sativa (Lata et al., 2016), Carthamus tinctorius (Vijaykumar et al., 2017), Pterocarpus marsupium (Ahmad and Anis, 2019), Tecoma stans (Hussain et al., 2019), Sesamum indicum (Elayaraja et al., 2019), Syzygium cumini (Naaz et al., 2019), Allamanda cathartica (Khanam et al., 2020), Maytenus emarginata (Shekhawat et al., 2021), Dioscorea pentaphylla (Manokari et al., 2022), Coleus forskohlii (Badhepuri et al., 2023), Ipomoea batatas (Behera et al., 2024) including the plants belongs to Solanaceae family like Withania somnifera (Kaur et al., 2021; Mahendran and Rahman, 2024), Physalis minima (Halder and Ghosh, 2021), and Solanum lycopersicum (Marapaka et al., 2024).
Synergistic effect of cytokinin and cytokinin on shoot induction
The cytokinins can induce several differentiation processes, such as shoot initiation, proliferation, and other developmental pathways (Petrasek et al., 2019; Phillips and Garda, 2019; Hurny et al., 2020). Among the three explants evaluated, cotyledon explants were more pronounced for both regeneration efficiency and production of the highest number of multiple shoot buds in the two genotypes. The cotyledon explants were used to find the most suitable combination and concentration of cytokinins. The medium was fortified with mT (8.28 µM), and different concentrations of cytokinins like BAP (2.22 to 8.88 µM), KIN (2.32 to 9.29 µM), and ZEA (2.28 to 9.12 µM) were added alone to determine the most appropriate combination of cytokinins in both genotypes. The addition of low concentrations of cytokinins induced an optimum regeneration response. The cotyledon explants generated the maximum number of shoot buds on the medium containing low concentrations of BAP, KIN, and ZEA (Table 4). The mT (8.28 µM) and BAP at 2.22 µM combinations generated 25.8 and 18.3 shoots in PPL and PGR genotypes, respectively (Table 4).
In contrast, the mT along KIN (2.32 µM) produced 20.4 and 16.4 shoots, and the ZEA (2.28 µM) had 21.3 and 16.6 shoots per explant in PPL and PGR genotypes, respectively (Table 4). The increased BAP, KIN, and ZEA concentrations significantly decreased the regeneration response and the number of shoots in both genotypes. The combination of mT and BAP was more efficient than the combination of ZEA and KIN regarding the number of shoots per explant and regeneration frequency for the three explants of two eggplant genotypes (Table 4). The combination two cytokinins is crucial for forming and increasing the number of shoots from different explants of various plants (Shekhawat et al., 2020). A synergistic effect of combining two cytokinins on the increased number of shoots in different plants has been observed, such as Carthamus tinctorius (Vijayakumar et al., 2017), Musa spp. (Mohapatra and Deo, 2019), Maytenus emarginata (Shekhawat et al., 2021), Gerbera jamesonii (Shaheen et al., 2022). The various combinations of two cytokinins, such as BAP + KIN, TDZ + BAP, and TDZ + KIN, were successfully employed and obtained the highest number of adventitious shoots in cotyledons of eggplant (Shivaraj and Rao, 2011; Foo et al., 2018; Yesmin et al., 2018). Our study confirms the earlier observation that the interaction of two cytokinins significantly enhanced the shoot initiation and proliferation efficiency in diverse explants of eggplant (Shivaraj and Rao, 2011; Bhat et al., 2013; Foo et al., 2018; Yesmin et al., 2018).
Synergistic effect of auxin and cytokinin on shoot induction
The interaction between cytokinins and auxins is essential in morphological development during in vitro initiation, multiplication, and proliferation stages. The developmental stages are mediated by the low auxin and increased concentrations of cytokinins by stimulating the initiation of buds, proliferation, and enhanced production of shoots (Phillips and Garda, 2019). The PGRs, explant, genotype, and other additives significantly affect the induction and proliferation of shoots and, subsequently, plant regeneration. The supplementation of varying concentrations of IAA (1.43 to 5.71 µM), IBA (1.23 to 4.92 µM), and NAA (1.34 to 5.37 µM) with mT (8.28 µM) determined their influence on the adventitious shoot induction and proliferation from all the explants of two eggplant genotypes. Among different auxins along with mT (8.28 µM), auxins at low concentrations (1.23 and 2.86 µM) were observed as efficient combinations for the maximum shoots in three explants of both cultivars (Table 5). The medium fortified with IAA (2.85 µM) and mT (8.28 µM) produced 26.4 shoots and 17.8 shoots in cotyledon explants of PPL (Fig. 1a) and PGR genotypes, respectively. In contrast, the other auxins like IBA (2.46 µM) amended with mT (8.28 µM) produced 22.4 shoots and 16.7 shoots, and NAA (2.68 µM) combinations with mT (8.28 µM) produced 21.6 shoots and 15.6 shoots in PPL and PGR genotypes of eggplant (Table 5). The media amended with more than (2.46 to 2.85 µM) of auxins showed no significant difference in the induction shoots in both genotypes.
The combination of auxin and cytokinin plays a significant role in different physiological processes mediating plant growth and development (Petrasek et al., 2019; Hurny et al., 2020; Hnatuszko-Konka et al., 2021). The auxin-cytokinin interactions are helpful in the development of shoot apex by synthesizing endogenous growth regulators, which reduce the exogenous supplementation of growth regulators (Gupta et al., 2020; Hurny et al., 2020). Multiple shoot buds were initiated on media containing different BAP concentrations with various auxins like IAA (Satish et al., 2015; Yesmin et al., 2018; Yarra and Kirti, 2019; Bhat et al., 2021), IBA (Satish et al., 2015), and NAA (Muktadir et al., 2016; Kaur et al., 2020) from various explants of eggplant cultivars. Several plant regeneration procedures have been demonstrated in various cultivars of eggplant using different explant types on media fortified with ZEA or Zeatin riboside (ZR) and three various auxins, namely IAA, IBA, and NAA individually (Pawar et al., 2013; Satish et al., 2015; Garcia-Fortea et al., 2020). The ZEA at 9.12 µM and IAA at 0.57 µM were the most suitable combinations for generating the optimal number of adventitious shoots in two eggplant cultivars (Muktadir et al., 2016). Similarly, the auxins in combination with mT show the synergistic effect on the shoot bud formation and proliferation in three explant types of two genotypes (Table 5). These studies are in accordance with the earlier studies on the auxin and cytokinin interaction, which significantly enhanced the number of shoots of other plant species like Tecoma stans (Hussain et al., 2019), Oxystelma esculentum (Jayaprakash et al., 2021), Dioscorea pentaphylla (Manokari et al., 2022), Coleus forskohlii (Badhepuri et al., 2023), Ipomoea batatas (Behera et al., 2024), and Solanum lycopersicum (Marapaka et al., 2024).
In this study, we have employed meta-Topolin to initiate adventitious buds from three explants of both eggplant cultivars. Among all the tested cytokinins, mT (8.28 µM) exhibited maximum adventitious shoots. The mT belongs to aromatic cytokinins, which occur naturally in plants (Aremu et al., 2012). The mT has been recorded as more suitable and efficient than BAP (Khanam et al., 2020). The mT contains a hydroxyl group at the aromatic side chain. This aromatic side chain helps to allow and accumulate O-glycosides (Lalthafamkimi et al., 2021), which are quickly metabolized and converted into active free bases. Whenever necessary, make available cytokinins for a longer duration for the initiation and increase the number of shoots in several plants (Erisen et al., 2020; Vylicilova et al., 2020; Jayaprakash et al., 2021; Lalthafamkimi et al., 2021). The mT has been observed as highly efficient in adventitious shoot initiation and proliferation compared to the other cytokinins BAP, KIN, 2iP, TDZ, and ZEA (Vijayakumar et al., 2017; Hussain et al., 2019; Khanam et al., 2020; Kucharska et al., 2020; Jayaprakash et al., 2021; Manokari et al., 2021; 2022). The mT has been used in several plants to improve regeneration efficiency, adventitious shoot induction, proliferation, and shoot quality and is involved in histogenic stability and physiological disorders (Koszeghi et al., 2014; Dimitrova et al., 2016; Kucharska et al., 2020; Jayaprakash et al., 2021; Manokari et al., 2021; 2022; Badhepuri et al., 2023).
Proliferation of multiple shoots and rooting
The elongation of adventitious shoot buds induced by different explants is a significant bottleneck, affecting complete plant regeneration. The adventitious shoots initiated by explants were unable to elongate. Hence, the explants with adventitious shoots were shifted to the medium fortified with low cytokinin concentrations after six weeks. The explants induced numerous shoot buds (< 3 mm in length counted with the help of a stereomicroscope) on varying combinations and concentrations of PGRs (Tables 1–5). The explants with clusters of tiny shoot buds were transferred to diverse combinations and media concentrations. The adventitious shoot bud clusters were cut into small bunches containing ~ 5–10 shoots shifted to the medium augmented with either individually or different combinations of mT (2.07 or 4.14 µM), BAP (2.22 or 4.44 µM), GA3 (0.29 or 1.45 µM), and IAA (0.57 or 2.86 µM) (Fig. 1d and e). Among diverse combinations and concentrations of PGRs, BAP at 2.22 µM alone or BAP (2.22 µM) and IAA at 2.86 µM were the most appropriate media for elongating shoots in eggplant. Our observations agree with the earlier results of the successful elongation of shoots in eggplant, where the low concentration of BAP enhanced the elongation of shoots (Sharma and Rajam, 1995; Khatun et al., 2022).
In contrast, the hormone-free medium was helpful in the shoot elongation from adventitious shoot buds initiated by various explants of eggplant (Mukhadir et al., 2016; Yesmin et al., 2018; 2021), and media with the same concentrations and combinations were used for initiation of shoots (Bhat et al., 2021). Different TIBA (Triiodobenzoic acid) and GA3 concentrations significantly increased the elongation of shoots induced by hypocotyl explants of eggplant (Mallya and Ravishanker, 2013). Similarly, the medium augmented with varying concentrations of GA3 exhibited a significant enhancement in the shoot elongation (Shivaraj and Rao, 2011; Kanna and Jayabalan, 2015). In the present study, mT at a low concentration (2.07 µM) successfully enhanced the elongation of shoots from the bunch of shoot buds initiated by different explants. Our observations confirmed the earlier results on the mT-induced proliferation of multiple shoots in various plant species like Tecoma stans (Hussain et al., 2019), Physalis minima (Halder and Ghosh, 2021), Oxystelma esculentum (Jayaprakash et al., 2021), Dioscorea pentaphylla (Manokari et al., 2022), Coleus forskohlii (Badhepuri et al., 2023), Ipomoea batatas (Behera et al., 2024), Withania somnifera (Mahendran and Rahman, 2024), and Solanum lycopersicum (Marapaka et al., 2024).
The elongated shoots obtained from different types of shoot induction media, such as mT and BAP, were evaluated for rooting efficiency. The rooting media are fortified with diverse auxin concentrations individually, such as IAA (1.43 to 5.71 µM), IBA (1.23 to 4.92 µM), and NAA (1.34 to 5.37 µM), used to determine the rooting frequency and suitable concentration of auxin and production of complete plantlets. The initiation rooting takes place within one to two weeks of culture. The root induction depends on the concentration and type of auxin employed in the medium. The diverse concentrations of auxins generate various degrees of rooting response and the number of roots per shoot in two cultivars of eggplant. The auxin concentration of more than 4.92 to 5.71 µM did not enhance the number of roots per shoot or the rooting efficiency of both eggplant cultivars. The medium supplemented with IAA at 5.71 µM was recorded as an appropriate auxin for the optimum number of roots (Fig. 1f and g). It enhanced rooting frequency in both eggplant genotypes, which showed a 99.4% rooting efficiency and initiated an average number of 13.6 roots/shoot in the PPL cultivar.
In contrast, 92.4% of rooting frequency and an average of 10.4 roots/shoot were recorded on the same medium in the PGR cultivar. The rooting frequency was 96.3%, and an average of 10.9 roots/shoot was shown in the PPL genotype on the rooting medium augmented with 4.92 µM of IBA, whereas 86.5% rooting efficiency and 7.4 roots per shoot were recorded in the PGR genotypes. The medium containing 5.37 µM of NAA induced 5.6 roots/shoot and 84.6% rooting efficiency, and 4.9 roots/shoot with 82.6% rooting frequency were shown in the PPL and PGR cultivars of eggplant. The IAA was found to be significantly more significant than other auxins (IBA and NAA) among the three auxins evaluated for rooting efficiency in both cultivars of eggplant. The best rooting response was exhibited in shoots of the PPL cultivar compared to the PGR cultivar. The auxin is critical in plant root meristem induction, proliferation, and other development processes (Gupta and Van Eck, 2016). The present study recorded IAA (5.71 µM) as a capable auxin type for initiating roots and improving rooting efficiency compared to IBA and NAA. Similar observations are reported as the IAA was the efficient auxin type for the proficient initiation of roots in eggplant (Satish et al., 2015). Meanwhile, IBA was the most competent auxin for inducing the rooting of shoots in many eggplant cultivars (Mallya and Ravishankar, 2013; Khatun et al., 2022). The shoots obtained from medium supplemented with mT show enhanced rooting efficiency compared to those generated from BAP-fortified medium. It was observed that the mT improves the rooting of shoots obtained from mT-supplemented media (Khanam et al., 2020; Shekhawat et al., 2021; Marapaka et al., 2024; Mahendran and Rahman, 2024).
Hardening and acclimatization
A total of 86 plants of the PPL genotype and 72 plants of the PGR genotype were placed under greenhouse conditions for acclimatization. Of these, 82 plants from the PPL genotype and 62 plants from the PGR cultivar were successfully acclimatized under greenhouse conditions (Fig. 1h). After four weeks of transplantation, a 95% survival rate for the PPL cultivar and a 90% survival rate for the PGR cultivar were observed. The plants produced from different explants are phenotypically similar to their mother plants. Our results on the maximum rate of survival confirm the competence of mT in enhancing the ability of acclimatization and rate of survival of regenerated plants as recorded in other plants (Khanam et al., 2020; Jayaprakash et al., 2021; Shekhawat et al., 2021; Marapaka et al., 2024).