High-frequency shoot regeneration from flower bud derived callus of Gymnostachyum febrifugum Benth., an endemic medicinal plant to the Western Ghats

Gymnostachyum febrifugum Benth. is a small, scapigerous, rare and endemic medicinal herb indigenous to India, and belonging to the family Acanthaceae. This study reports an efficient protocol for high-frequency flower bud-derived callus induction and shoot organogenesis in G. febrifugum. Flower buds at 7 days before anthesis (dBA) were excised from the inflorescence and cultured on MS medium supplemented with various concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 2.275–9.1 µM) for callus induction. The optimum callus induction (78%) was obtained on MS medium supplemented with 6.825 µM 2,4-D. The calli when subcultured on MS medium supplemented with different concentrations of thidiazuron (TDZ; 2.275–11.375 µM) or 6-benzylaminopurine (BAP; 2.22–11.1 µM) alone or in combination with 1- naphthaleneacetic acid (NAA; 1.074–3.759 µM) induced shoots. The highest frequency (94%) and number of shoots (44.6 shoots/unit callus) were obtained on MS medium supplemented with 9.1 µM TDZ and 2.685 µM NAA. The optimum rooting frequency (95%) and number of roots (10.2) were observed on ½ MS medium supplemented with 14.76 µM indole-3-butyric acid (IBA). The rooted plantlets were acclimatized and transferred to soil with 94% success. A protocol for high-frequency shoot induction from flower bud derived calli has been standardized for G. febrifugum,an endemic medicinal herb to the Western Ghats.


Introduction
Gymnostachyum febrifugum Benth. is a rare endemic plant belonging to the family Acanthaceae (Thomas et al. 1996). This is a pretty, stemless, tiny and scapigerous herb with large long-petiolated ovate leaves and woody rootstock ( Fig. 1; Gopalakrishna Bhat 2014). This plant is indigenous to India and has an attractive flower, which makes it a potential wild ornamental plant (Thomas et al. 1996).
The inflorescence in G. febrifugum is a spike with limited flowered cymes and the flower is white tinged with purple with a yellow lower lip. The root of this plant is used as febrifuge and against blisters and sores on the tongue (Pattanayak 2019).
According to Mascarenhas (2010), the threat status of this plant is near threatened. Hence, there is an urgent need for the conservation of this plant. Conventional propagation of this plant is through seeds and our study revealed that the seed formation in fruits is relatively low probably due to self-incompatibility. Additionally, low viability and formation of a high percentage of sterile seeds are the difficulties associated with its propagation. Further, vegetative propagation is not very promising due to the lack of erect stems in G. febrifugum. In this background, micropropagation is considered as a viable option to rapidly propagate this plant (Ramírez-Mosqueda and Iglesias-Andreu 2016).
Since this plant is devoid of an erect stem, the collection of explants such as leaf and subterranean parts often cause high rates of contamination. The flower bud is considered as an ideal explant for micropropagation of this plant since it can reduce the contamination rate substantially and also avoids destroying the mother plant. However, the collection of flower bud explant is limited only during the flowering period.
Although plant tissue culture related instability in regenerating plants is prevalent in several systems, somaclonal variation is considered as an additional tool for the improvement of agricultural as well as horticultural crops along with well-established plant breeding methods (Arun et al. 2003;Santha and Mehta 2001;Anil et al. 2018). The cause of such variations is essentially due to various reasons such as genetic, phenotypic and epigenetic alterations observed in plants growing in cultures (Karp 1995). Such variations may increase if the pathway is through callus organogenesis. Other factors which determine the variations include the medium and maintenance time in culture. The somaclonal variants usually appear due to the structural and numerical changes in chromosomes (Bairu et al. 2011;Delgado-Paredes et al. 2017).
The use of plant tissue culture techniques has been considerably increased over the past few decades for the mass propagation of rare, endangered, endemic and threatened plants (for review see Deepa and Thomas 2020). Micropropagation techniques can give rise to large number of plants in a very limited period irrespective of season and climate (Liao et al. 2011). This technique is more effective in producing mass scale elite, healthy, disease-free plants compared with that of conventional propagation protocols (Vieitez et al. 2007;Corredoira et al., 2017). Further, this technique can support the improvement of agriculture, horticulture and forestry plants. Various approaches such as nodal segment culture, adventitious shoot formation, direct and indirect organogenesis and somatic embryogenesis are frequently used for the multiplication of various plant species (Demétrio et al. 2021;Kannan et al. 2021;Saavedra et al. 2021;Patel et al. 2021). To our knowledge, there is no report on in vitro propagation of G. febrifugum. Therefore, in this study, we report an efficient high frequency shoot induction protocol from flower-bud derived callus.

Plant material and surface sterilization
Gymnostachyum febrifugum Benth. was collected from Periya, Kasaragod district in Kerala. Authentication was carried out by the expert Dr. A. K Pradeep and the voucher specimen (No. CU 7060) has been submitted in the Calicut University herbarium, University of Calicut, Kerala, India.
G. febrifugum flowers from July to November. The plant bears beautiful flowers during this period. The flowers are arranged in a cymose inflorescence. Flower buds were collected from such inflorescences ( Fig. 2A). For our study, we have selected flower buds at three different stages of development i.e. 4, 7, 10 days before anthesis (dBA). The flower buds were excised from the inflorescence of fieldgrown plants and brought to the lab in the morning. Each flower bud has a small stalk at the cut end (Fig. 2B). The flower buds were washed thoroughly in sterilized distilled water for 5 min and then immersed in sterilized distilled water containing 1% Savlon solution for 8 min. followed by two rinses in sterilized distilled water. Finally, the explants were surface sterilized with 0.1% mercuric chloride (HgCl 2 ) for 7 min. The explants were then washed three times with sterilized distilled water. The buds were then kept in sterilized glass Petri plates lined with blotting paper to remove traces of water.

Media and culture initiation
Murashige and Skoog medium (MS medium; Murashige and Skoog 1962) was invariably used throughout the experiment and is supplemented with various plant growth regulators. The pH of the medium was adjusted to 5.8 by using 0.1 N NaOH or 0.1 N HCl before gelling with 8.0 g/l bacteriological agar (Himedia, India). For callus induction from flower buds, different concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D; 2.275-9.1 µM) were employed. The flower buds were placed horizontally in Petri plates and the basal stock portion of the flower buds was slightly pushed in the medium at the time of inoculation (Fig. 2C). The calli induced from explants were subcultured on MS medium supplemented with 2.275 µM 2,4-D for multiplication every 4 weeks.

For shoot regeneration from callus
The callus was subcultured on MS medium augmented with various concentrations of thidiazuron (TDZ; 2.275-11.375 µM) or 6-benzylaminopurine (BAP; 2.22-11.1 µM) alone and in combination with 1-naphthaleneacetic acid (NAA; 1.074-3.759 µM) for shoot induction. Each container was inoculated with 1.0 g callus. The percentage response, number of shoots and mean shoot length were measured 45 days after culture.

Culture conditions and statistical analysis
The cultures were incubated at 22 ± 2 °C, in the culture room with 16-h light conditions and 40-50 μmol m 2 /s photosynthetic photon flux (PPF) provided by white,cool fluorescent lamps. For each experiment, at least 14 replicates were taken and each experiment was repeated three times. Statistical analysis was performed by using analysis of variance (ANOVA) through SPSS software. Data were analysed for the significance of differences of means among treatments using Duncan's multiple range test (DMRT) at P ≤ 0.05.

Callus induction from flower buds
In almost all concentrations of 2,4-D tested, calli with similar textures were induced whereas on basal medium (control) there was no callus induction (Table 1). Callus initiation started 2 weeks after culture (Fig. 2D). Initially, the calli appeared as small granules on the surface of the explant and later they slowly spread to the entire surface. After 6 weeks the calli completely covered the explants in responding cultures (Fig. 2E). Due to the presence of anthocyanin pigments in the explant, the calli showed pink spots occasionally (Fig. 2F). Table 1 depicts callus induction using flower buds at 4, 7 and 10 days before anthesis (dBA) on MS medium supplemented with 2.275, 4.55, 6.825 and 9.1 µM 2,4-D 45 days after culture. There was no callus induction on MS basal medium. The percentage explants with calli and callus size were influenced by the age of the flower bud as well as concentration of 2,4-D. Seventy eighty percentage of explants produced large calli while cultured on 6.825 µM 2,4-D when flower buds were collected 7 dBA (Table 1). On this medium, the earliest callus induction was noticed (7-9 days) and flower buds from 4 and 10 dBA took longer to induce callus (12-14 days). All the calli induced were of friable texture. Since the highest callus induction frequency and callus size was observed on 6.825 µM 2,4-D in flower buds collected 7 dBA, all further callus induction experiments were done with this explant and media combination. The age of the flower bud is an important factor, which influences callus induction and subsequent shoot organogenesis in some systems. In Gerbera jamesonii 7, 8, 9, 10 and 11-days-old flower buds were cultured to induce callus. In all four varieties, 7-9-days-old flower buds showed the best response in terms of both callus induction as well as shoot organogenesis (Akter et al. 2012). The auxin 2,4-D is routinely used for callus induction from various explants in different systems (Rathore et al. 2011;Abraham and Thomas 2015;Bala et al. 2015;Patricia et al. 2021). According to Zheng and Konzak (1999) the largest 2,4-D-induced callus induction could be achieved not only by using the optimum concentration but also its duration of presence in the medium. In this study, we obtained optimum callusing at 6.825 µM 2,4-D. Ozias-Akins and Vasil (1982) found that 2,4-D up to 9.1 µM was found favourable for callus induction and beyond this concentration inhibited the cell division in wheat.
The calli during the initiation stage remained predominantly pinkish in colour whereas as the calli grew further, they became light yellow and green (Fig. 2D-F). However, pinkish spots were regularly noticed on all calli during the entire period of culture. This pinkish colour is due to the exudation of the flavonoid compound anthocyanins present in the explants. The presence of anthocyaninswas reported in various parts of some plants especially in flowers, fruits and vegetables (Mihai et al. 2010). The induced calli were subcultured on MS medium supplemented with 2.275 µM 2,4-D for multiplication after 45 days.

Shoot regeneration from callus
Well-developed friable yellowish-white calli with pink spots were subcultured on MS medium supplemented with TDZ (2.275-11.375 µM) or BA (2.22-11.1 µM) alone or in combination with NAA (1.074-3.759 µM) for shoot induction ( Table 2). The calli were inoculated (2.0 g fresh weight per bottle) in culture bottles containing 150 ml medium. In this study, two cytokinins were used for shoot organogenesis from callus i.e. BAP and TDZ. The response was low when these cytokinins were used individually. However, the  (Table 2). Although the percentage response and number of shoots varied depending on the type of plant growth regulators and concentrations, there was not much difference in mean shoot length. Some regions of the calli turned green after 2 weeks of culture and shoot initiation started from such regions (Fig. 2F). Later the shoots were formed all over the surface of the calli (Figs. 2G, 3A). In the present study, a combination of TDZ and NAA produced the highest shoot induction from callus. In cabbage, TDZ in combination with NAA was found superior over combination with other auxins in inducing shoots (Gambhir and Srivastava 2015). The synergistic action of TDZ and auxin-induced shoot induction has been reported in several systems including Solanum khasianum (Chirumamilla et al. 2021), Ficus carica (Abdolinejad et al. 2020) and Dieffenbachia (Shen et al. 2008). TDZ is an active cytokinin extensively used for shoot induction from various explants in different systems (Deepa et al. 2018). TDZ was found more efficient than other cytokinins in some systems (Zhang et al. 2011;Bhattacharyya et al. 2016). TDZ stimulated shoot organogenesis from various systems including medicinal plants, trees and endangered plants and has been reported by many workers (for review see Deepa et al. 2018). TDZ can not only stimulate the metabolism of other PGRs but also it can increase auxin accumulation, translocation and cytokinin content (Casanova et al. 2004;Kodja et al. 2015).
In the present study, we used the flower bud as explant. There are various reports in which young tissues such as flower buds and inflorescence segments were used as explants for shoot induction. In Arnebia hispidissima of the various explants such as leaf, immature inflorescence and mature and immature seeds, employed for callus induction and shoot regeneration, only the immature inflorescence responded (Phulwaria and Shekhawat 2013). Flower buds were used for micropropagation in some systems (Akter et al. 2012;Liao et al. 2011;Yang et al. 2011). Young tissues such as immature inflorescence, flower buds have high meristematic activity and form excellent material for micropropagation studies (Kackar and Shekhawat 1991;Yadav et al. 2009;Głowacka et al. 2010). There are some reports, which suggest that floral structures have more ability to reproduce vegetatively than other plant parts (Gingas 1991;Lopez-Baez et al. 1993;Merkle et al. 1997;Steinmacher et al. 2007).

Rooting of shoots
For root induction, micropropagated shoots measuring a size of about 1.0 cm or above were used. The shoots were cultured on ½ MS medium supplemented with various concentrations of indole-3-butyric acid (IBA; 4.92-19.68 µM). On MS basal medium (control) there was no root induction (Table 3). Half strength MS medium supplemented with 14.76 µM IBA showed the highest frequency of root induction (95%) and number of roots per explant (10.2; Fig. 3B) as well as mean root length (2.6 cm). IBA induced root induction has been reported in other systems such as Plumbago zeylanica (Sharma and Agarwal 2018), Pterocarpus marsupium (Ahmad et al. 2021) and Swertia minor (Kshirsagar et al. 2021). The rooted shoots were acclimatized successfully and transplanted to soil (Fig. 3C).
In conclusion, a robust shoot regeneration system through flower bud-derived callus organogenesis has been standardized for G. febrifugum. From this study it is clear that flower-bud is an ideal explants for plant regeneration in G. febrifugum. The addition of 2,4-D in the medium supported callus proliferation from explant. Synergestic 22.9 ± 2.6 cd 1.0 ± 0.05a 6.66 2.685 55 ± 9.1d 28.4 ± 2.8bc 1.1 ± 0.04a 6.66 3.759 51 ± 8.7d 25.3 ± 2.5c 1.2 ± 0.08a effect of auxin and cytokinin induced more shoots from callus than cytokinin alone. A combination of TDZ and NAA produced optimum shoot organogenesis in G. febrifugum. This protocol could be utilized for the mass multiplication of this plant for commercial purposes.