Micropropagation of Andrographis producta through axillary and adventitious shoot regeneration
Journal of Genetic Engineering and Biotechnology volume 20, Article number: 152 (2022)
Andrographis producta (C. B. Clarke) Gamble is a valuable medicinal plant that yields several therapeutic compounds. In addition, this species is endemic to the Western Ghats regions of South India. Natural populations of Andrographis producta have dwindled due to the overexploitation of this species. The objective of the present study was to develop a reliable In vitro propagation protocol for this plant species.
In vitro plant regeneration protocol has been developed in Andrographis producta using nodal and shoot tip explants. The highest axillary shoots (14.60) were regenerated from nodal explants on MS medium amended with 10 μM 2-iP. Similarly, on MS media amended with 5 μM BAP, 17.50 shoots were regenerated from shoot tip explants. Optimal of 27.66 adventitious shoots were regenerated from the cut end of shoot tip explants on MS medium amended with 10 μM 2-iP. Medium amended with 10 μM 2-iP was optimum for regeneration of multiple axillary shoots from nodal explants and for the adventitious shoots regeneration from shoot tip explants. Shoot tips were ideal explants for the micropropagation of A. producta. Qarter sthrength MS media supplemented with 10 μM IBA has resulted in maximum rooting of the shoots.
A reliable In vitro micropropagation method was developed in Andrographis producta through direct organogenesis, and this method is helpful for the multiplication, conservation, and utilization of this plant.
Andrographis (Acanthaceae) comprises threatened and endemic medicinal plants such as A. producta, A. alata, A. lineata, and A. echioides distributed in the South Indian region [1,2,3,4]. In contrast, the A. paniculata is a regularly grown plant in India. It has been produced in Southeast Asian countries like China, India, and Malaysia to meet the demand for both traditional and modern medicinal systems . Andrographis species contain diterpenoids, flavonoids, and xanthones which are reported to possess a wide range of biological properties such as anticancer, antidiabetic, anti-inflammatory, antimicrobial, antioxidant, immunostimulant, and hepatoprotective activities .
Andrographis producta (C. B. Clarke) Gamble is an erect herb that is endemic to the Western Ghats in India . Andrographis producta is reported to possess phytochemicals, including phenolics, flavonoids, and organic acids, which have shown potent antioxidant activities . Gas chromatography and mass-spectroscopic analysis have demonstrated the presence of various bioactive compounds such as 2-methoxy-4-vinylphenol, 2,4-ditert-butyl-phenol, phytol, 5-hydroxy-7,8-dimethoxyflavone, gammasitosterol, salvigenin, solanesol, and alpha-terpinene in this plant . Andrographis producta is traditionally used to treat various health ailments, including intestinal worms, relieve constipation, eliminate phlegm in womens during postpartum by native people , and treat skin diseases by the local tribes of Nilgiris Biosphere Reserve . Endemism, habitat loss, forest fires, and overexploitation are significant threats to the survival of Andrographis species . Therefore, plant regeneration protocols have been developed for A. paniculata , A. alata , A. macrobotrys , A. echioides , and A. lineata . In addition to the facts mentioned above, conventional propagation of Andrographis producta through seeds is hampered by poor seed germination and short seed viability [15, 16], and there are no regeneration protocols for the micropropagation of Andrographis producta. Given the above, the In vitro propagation method was adopted for the large-scale production of Andrographis producta plants. Here, we report successful methods for large-scale propagation using nodal and shoot tip explants.
Plants of Andrographis producta (C.B. Clarke) Gamble were collected from Bababudan Hill ranges, Chikamagalur district, Karnataka, India (lat: 13° 25′ 10.2108″; long: 75° 44′ 37.0026″; MSL 1467.30 m) and were maintained in a botanical garden. Identification of plant species was confirmed by Prof. S. R. Yadav, Shivaji University, Kolhapur, India, and voucher specimens were maintained at herbarium, Shivaji University, Kolhapur, India.
In vitro seed germination
Seeds were sterilized with 5% (v/v) sodium hypochlorite solution for 15 min, cultured on 1/10th-strength Murashige and Skoog  (MS) medium supplemented with 3% sucrose, and solidified with 0.8% agar and incubated in culture room at 25 ± 2 °C with a photoperiod of 16/8 h (light and dark). All processes were carried out under sterile conditions using a laminar air-flow hood. In addition, the pH of the medium was set at 5.8 and sterilized by autoclaving at 121 °C for 15 min.
Shoot tip and nodal cultures
Shoot tips (1–3 mm) and nodal explants (5 mm in length) were obtained from 6-week-old seedlings. They were cultured on MS nutrient medium supplemented with 3% (w/v) sucrose and 2.5, 5.0, 7.5, and 10.0 µM BAP, KN, 2-iP, and TDZ (HiMedia, India) individually. The cultures were maintained in culture room wherein temperature, light, and relative humidity were set at 25 ± 2 °C, 16 h light (50 µmol m−2 s−1)/8 h dark, and 60% relative humidity, respectively.
In vitro root formation
Regenerated shoots were individually cultured onto ¼ strength MS nutrient medium containing 3% (w/v) sugar, supplemented with 1.0, 2.0, 5.0, and 10 µM IAA, IBA, and NAA (HiMedia, India) for induction of roots.
Acclimatization of plants
Micropropagated plants (5 cm in height) were transplanted to pots containing equal volumes of cocopeat and vermiculite and plants were reared in growth chambers wherein temperature, light, and relative humidity were set at 25 ± 2 °C, 16 h light (50 µmol m−2 s−1)/8 h dark, and 60%, respectively. After 2 weeks, plants were transferred to potting mix containing soil and cocopeat and maintained in the greenhouse.
For histological studies, cultured nodal and shoot tip explants were fixed in FAA (10 ml of formalin, 85 ml of 70% ethyl alcohol, and 5 ml of glacial acetic acid) for 12 h at room temperature and dehydrated by ethanol-butyl alcohol series and embedded in parafilm as recommended by Johansen . The material was sectioned (thickness of 6 µm) and stained with 0.05% toluidine blue (HiMedia, Mumbai, India) and examined under a compound microscope (Nikon, Tokyo, Japan).
A randomized block method was followed for the establishment of experiments. Data were statistically analyzed with the help of one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was applied using SPSS statistical software (version 20).
Axillary shoot regeneration from nodal explants
The nodal explants cultured on MS medium containing cytokinin involved in axillary shoot induction within 2 weeks of culture (Table 1). Axillary shoots were regenerated on all cytokinin-supplemented media; however, optimum regeneration was observed on MS medium supplemented with 2-iP. The highest percentage of response, and a greater number of shoots, and mean shoot length were optimum with 2-iP containing medium (Table 1). On 2-iP containing media, initially, few shoots were emerged from nodal regions after 2 weeks in culture (Fig. 1A). Shoot proliferation was evident with the advancement of time (after 4 and 6 weeks) in culture (Table 1, Fig. 1 B–C); at the end of 8 weeks in culture, highest shoot proliferation was recorded (Fig. 1D). On MS media amended with 10 μM 2-iP, the highest number of 14.60 shoots were regenerated from nodal explants (Table 1). Nodal explants involved in direct shoot regeneration without callus phase or callus mediation were examined histologically (Fig. 2A).
Multiple and adventitious shoot regeneration from shoot tip explants
Seeds were germinated on 1/10th strength MS basal medium (Fig. 1E), and shoot tips harvested from young seedlings were used for plant regeneration. Shoot tip explants cultured on MS medium amended with KN and TDZ (2.5, 5.0, 7.5, and 10 μM) developed a single shoot. However, the shoot tip explants cultured on BAP and 2-iP media showed a differential response. On media supplemented with BAP, shoot tip explants developed multiple shoots from shoot meristem (Table 1). Shoot tip explants initially produced a few shoots (Fig. 1F), and shoot proliferation was observed after 4 and 8 weeks in culture (Table 1; Fig. 1G–H). On MS media amended with 5 μM BAP, 100% shoot tips were responded and developed 17.50 shoots per explant (Table 1; Fig. 1H). Shoot tip explants which were cultured on 2-iP containing media showed adventitious shoot regeneration from the cut end of shoot tips. In contrast, a single shoot was regenerated from the shoot meristem on basal medium. Shoot buds sprouted from the cut end of the shoot tip on MS medium containing 10 μM 2-iP after 2 weeks in culture (Fig. 1I); such shoot buds were involved in proliferation in subsequent weeks (Fig. 1J–K). Optimal of 27.66 adventitious shoots were regenerated from the cut end of shoot tip explants on MS medium amended with 10 μM 2-iP (Table 1). Preexisting meristem of shoot tip explants cultured on 5 μM BAP divided and differentiated into multiple shoots, and shoot meristems directly generated multiple shoots without callus tissue’s mediation, according to histological preparations of shoot tip explants (Fig. 2B). Similar to this, histological studies revealed direct adventitious shoot regeneration (without callus intervention) from the cut end of shoot tip explants as a result of mitotic activity of epidermal cells in response to the media supplemented with 10 μM 2-iP (Fig. 2C).
In vitro root formation
For root induction, In vitro raised shoots (2-5 cm in length) were cultured on quarter strength MS media amended with 1, 2, 5, and 10 μM IAA, NAA, or IBA, and the results are presented in Table 2. Roots were sprouted from the shoots on all the auxin-supplemented media (Table 2); however, optimal root induction was recorded on MS medium containing IBA. There was a linear increase in the number of roots with the increasing concentration of IBA in the medium (Table 2, Fig. 3A). The highest percent of root induction (100%) and optimum roots per shoot (18.66 per shoot) were observed on MS medium supplemented 10 μM IBA (Table 2, Fig. 3A).
In vitro regenerated plantlets were removed from culture vessels and media adhering to the roots of the plantlets was carefully washed with distilled water. The plantlets were transplanted to poly-cups containing cocopeat and vermiculite (1:1 ratio) and reared in controlled conditions for 2 weeks (Fig. 3B). Later, they were transferred to bigger pots containing potting mix (Fig. 3C), and the survival percentage was 95%.
The present In vitro propagation studies reveal that multiple axillary shoots could be regenerated by using nodal explants of A. producta on MS medium supplemented with 2-iP. In contrast, media supplemented with KN, BAP, and TDZ were less efficient in multiple axillary shoot regeneration. On MS media amended with 10 μM 2-iP, the highest number of multiple shoots were regenerated from nodal explants. Additionally, histological examination showed that nodal explants directly produced shoots without the need for callus mediation (Fig. 2A), and the similar reports on use of histological evidences to trace the mode of regeneration from nodal explants have been reported in Vitex trifolia , and Andrographis paniculata , where meristematic cells at outer protoderm layer of axillary bud divided and differentiated into axillary shoots. Multiple axillary shoot regeneration from nodal explant on cytokinin supplemented medium was also reported in Andrographis paniculata, Artemisia nilagirica var. nilagirica, Artemisia japonica, Feronia limonia, Nothapodytes nimmoniana, Spilanthes oleracea, and Vitex trifolia [10, 19,20,21,22,23,24]. Among various cytokinins used in the present study, 2-iP supplemented medium was superior in axillary shoot induction. In contrast to the present results, BAP was reported to be potent cytokinin in axillary shoot induction in Andrographis alata and A. macrobotrys [11, 12].
Shoot tip explants of Andrographis producta developed multiple shoots from shoot meristem on MS media amended with 5 μM BAP. In contrast, the development of adventitious shoots was recorded from the cut end of the shoot tip explants (the basal portion of the shoot tips) on media amended with 2-iP. Optimal of 27.66 adventitious shoots were regenerated from the cut end of shoot tip explants on MS medium amended with 10 μM 2-iP. Histological preparations again showed direct regeneration of multiple shoots from the shoot meristem and basal portion of the shoot tips, and this is in consistent with the reports on Clementis cultivar where meristems of shoot tip are involved in division and differentiation to form direct shoots  and Neolamarckia cadamba , where epidermal and subepidermal cells regained mitotic activity and differentiated directly into new shoots. Among the four individual cytokinins tested, i.e., KN, BAP, TDZ, and 2-iP, the highest multiple shoot regeneration was achieved on 2-iP supplemented medium. Similar to the current results, an efficient In vitro plant regeneration was achieved from shoot tips explants of Curculigo latifolia  and Enicostema axillare  on TDZ and BAP supplemented medium, respectively. Adventitious shoot regeneration from shoot tip explants was described in Vanda coerulea by Jitsopakul et al.  which was the efficient mode of In vitro regeneration.
An essential stage for plantlet regeneration and adaptation is the rooting of an In vitro regenerated shoot. A single shoot of Andrographis producta cultivated on quarter strength MS media supplemented with 10 M IBA resulted in the optimum root induction. After 2 weeks of root initiation, there was a rapid root elongation. Andrographis paniculata , Andrographis alata , and Andrographis macrobotrys  all showed comparable results.
The MS media with 10 μM 2-iP was proved as most effective for the adventitious shoot and axillary shoot induction from shoot tip and nodal explants, respectively. For rooting, qurater sthrength MS media amended with 10 μM IBA has resulted in maximum rooting. This study showed an efficient direct shoot regeneration of Andrographis producta using nodal and shoot tip explants. The In vitro regeneration protocol developed for Andrographis producta is useful for the multiplication and conservation of this plant.
Availability of data and materials
α-Naphthalene acetic acid
Murashige and Skoog medium
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Kadapatti, S.S., Murthy, H.N. Micropropagation of Andrographis producta through axillary and adventitious shoot regeneration. J Genet Eng Biotechnol 20, 152 (2022). https://doi.org/10.1186/s43141-022-00438-w