Effect of Plant Growth Regulators and Growth Additives on the In vitro Micropropagation of Aerides odorata Var. Alba from Immature Pod Seeds

North-eastern Indian state of Manipur is renowned for having the fourth-highest orchid diversity in the world (Medhi and Chakrabarti, 2009). Among the 251 species of wild orchids identified in Manipur, Aerides is a valuable commercial medicinal orchid (Paraste et al., 2023). A. odorata is a wild species of Thailand, an epiphytic orchid with monopodial growth habit. It is collectively referred to as a Fox-tail orchid because of its flowered bush-like inflorescence (Hongthongkham and Bunnag, 2014). The flowers of A. odorata Lour. starts blooming usually in May-July (Sangma et al., 2021). It has a pleasant odour and a white blossom with a pinkish tinge (Al-Faruque  et al., 2015). Their scent has made them a valuable source for the scent extraction and production of numerous artificial hybrids and cultivars. They may be used in the perfumery and pharmaceutical industries. The leaves of A. odorata Lour. also has antibacterial properties. It has been shown to possess alkaloids and other naturally occurring chemicals with various antibacterial, antioxidant and anticancer activities (Paraste et al., 2023). The fruit of this orchid is mainly use for the treating of wounds by traditional healers of Manipur, while the juice from the fleshy leaves of this orchid is used for healing of boils in nose and ear. Due to its various medicinal uses, there is an indiscriminate wild collection and illegal trade by the people which has led to the loss of its biodiversity and urges to initiate various conservation methods to prevent this orchid from becoming extinct. But one of the challenges in conserving this species is their prolonged and complex propagation process. The propagation of this species through sexual means is a very slow process as its seeds lack endosperm. The size of orchid seeds can vary significantly (Akhila et al., 2024) and need fungal stimulant for germination in nature to provide the necessary physiochemical stimuli for seed germination and development (Lal et al., 2020). The fungus is believed to augment the carbohydrate, auxin and vitamin transport in the orchid (Arditti et al., 1982).  Hence, the aims of the present study were to investigate the suitable medium for seed germination and the suitable concentration of plant growth regulators to establish plantlets from A. odorata seeds via in vitro culture.

Preparation of explant
 
The immature and un-dehisced pods of A. odorata var. Alba was acquired from M/s Kwaklei and Khonggunmelei Orchids Private Limited, Imphal, Manipur, during July 2021. The research investigation was conducted in the Tissue Culture laboratory at the Department of Genetics and Plant Breeding, Central Agriculture University (CAU), Imphal, Manipur. The orchid pods were soaked with Tween 20 (0.01%) in a shaker for 30 to 40 min and washed vigorously for 15 to 20 min under running water. Inside the Laminar airflow hood, they were dipped in 70% ethyl alcohol for 30 sec, flamed and sterilized for 8 min within a solution containing Mercuric chloride (HgCl₂) (0.1%) and 1 to 2 drops of Tween 20. They were rinsed with sterilized distilled water and the pods were treated with Streptomycin (0.02%) and Bavistin (0.01%) solutions for 6 to 7 min each. After that, they were repeatedly washed 5 to 6 times with sterilized distilled water to eliminate all sterilizing agent remnants. Lastly, the sterilized capsules were sliced open longitudinally with a sterilized blade in an aseptic setting inside a laminar airflow cabinet to expose the immature seeds.
 
Seed viability test
 
Tetrazolium chloride was used to determine whether the seeds were viable. After soaking in sterile water for 24 h, the seeds were treated with a final application of 1% (w/v) solution of 2, 3 and 5 triphenyl tetrazolium chloride (TTC) (HiMedia, Mumbai, India) before being immersed in a solution of Calcium oxy-chloride (Ca(OCl₂)), 5% (W/V) and Tween 20, 1% (V/V).  The Petri dish was immediately placed in a dark room for around 12 hours and the seeds were examined in a microscopic field. The non-viable embryos remained colourless, whereas the viable ones became red.
 
In vitro seed germination
 
The seeds were inoculated on ½ MS media augmented with different concentrations of BAP viz., 0.1, 0.5, 1.0 mg/L (HiMedia, Mumbai, India) and 15% coconut water (HiMedia, Mumbai, India) to enhance seed germination and subsequent development. Then, 3% sucrose (HiMedia, Mumbai, India) was added and gelled by using 0.8% (w/v) agar. The pH of the medium was adjusted to 5.8±0.2 either with 0.1 N NaOH or HCl before autoclaving at 121^oC and 151 psi for 15 to 20 min. The cultures were kept at a temperature of 25±2^oC and exposed to 16 h, using fluorescent tubes (40w Philips India Ltd., Mumbai) by illuminating 3500 lux intensity of light. In each treatment, 100 seeds were cultured in culture phytajars (200 ml, HiMedia, Mumbai, India) filled with 50 ml of the medium. All treatments were triplicated with 4 culture phytajars in each replication.

The rate of germination was estimated using the formula:
 

   

 
The observations were recorded accordingly.
 
In vitro shoot formation from seed using different plant growth regulators (PGRs)
 
Different concentrations of NAA (2.0 mg/L) and BAP (0.1-4.0 mg/L) were added to ½ MS medium for shoot induction. After autoclaving, the media was maintained at 25±2^oC temperature and exposed to 16 hours of normal light followed by 8 hours of dark. The observations were recorded for 15 days until the 60^th day after inoculation.
 
In vitro rooting of regenerated shoots using growth regulator and additives
 
The shoots obtained from the seed germination were used for inducing roots. The regenerated multiple shoots were excised and single shoots particularly longer than 1cm with 1-2 leaves were cultured individually on ½ MS media supplemented with various growth regulators viz., NAA, IBA, IAA (0.5 mg/L) and growth additive, banana powder (50 g/L). The cultures were maintained in the culture room under the same conditions as done above. The observation was recorded at regular intervals accordingly.
 
Statistical analysis
 
The experiment was conducted in completely randomized design (CRD). The significance and non-significance of the variance due to different treatments were determined by calculating the respective ‘F’ values as the method described by Gomez and Gomez (2010).

In vitro seed germination
 
The effects of different concentrations of PGRs and growth additives on orchid seed germination indicated a positive response. Out of the eight treatment combinations, higher seed germination was observed with T7 which involved a medium of ½ MS augmented with 2.0 mg/L BAP and 15% Coconut water. The swelling of the seed was the first visible change observed in the explants followed by the development of a swollen spherical green corm-like embryo i.e., Protocorm-like body (PLB) (Fig 1) which are structurally similar to protocorms and arise from the explants (Gantait et al., 2020). PLBs initially broke through the fine membranous seed covering and developed a fine layer of rhizoids during the typical germination process which eventually developed into a seedling. Seed germination was noticed within just 25.50 days of inoculation in T7. Meanwhile, a delay in seed germination was observed in T1 (43.42 days). These differences in the rate of germination clearly illustrate the efficiency of the different treatment combinations. These findings corroborate several prior studies such as those of Prarab and Krishnan (2012) in Aerides maculosa Lindl. and Rhynchostylis retusa (L.) Bl; Vijayakumar et al., (2012) in Dendrobium aggregatum Roxb. and Piri et al., (2013) in Acampe papillosa (Lindl.) Lindl where they also observed accelerated germination rates in response to specific growth additives like 15% coconut water. Coconut water is renowned for being a rich source of essential minerals, amino acids, sugars, antioxidants, organic acids and most importantly, growth regulators like cytokinins which significantly spur in vitro orchid growth (Yong et al., 2009). Their role in seed germination is crucial as they enable critical processes such as cell division, thereby expediting the onset of germination.


       
Furthermore, the highest rate of germination was spotted in T7 (Table 1), where 72.42% of the seeds germinated after the 60th day of inoculation. It was substantially larger than the control group, where only 20.50% of the seeds had achieved germination. Introducing PGRs to the media improves orchid seed germination and seedling growth. According to Dohling et al., (2010), BAP additions increased the frequency of Dendrobium formosum Roxb. ex Lndl germination. The stimulatory impact of BAP on protocorm multiplication and shoot formation in Cymbidium pendulum (Roxb.) was also documented in the investigations carried out by Pathak et al., (2001). Growth may be boosted by combining PGR and growth additive, however, the effects of these mixtures depend on the type of orchid and the growth regulators that are employed, as well as their combination and ratios.


 
In vitro shoot formation using different PGRs
 
In the present study, different concentrations of PGRs viz., BAP and NAA in six combinations and a Control used for studying shoot initiation revealed that with increasing BAP and NAA concentrations, the frequency of shoot regeneration and the total number of shoots per explant increased (Table 2). Earliest shoot initiation (17.17 days), highest number of shoots (6.58), longest shoot (2.18 cm) and highest number of leaves per explant (10.00) were observed at ½ MS media supplemented with high concentrations of BAP (4.0 mg/L) and NAA (2.0 mg/L). PGRs play a vital role in regenerating cell differentiation, the combined treatment of BAP and NAA is found to have a positive effect on shoot growth and multiplication as reported in A. multiflora Roxb. by Bhowmik and Rahman (2020) and Devi et al. (2013) in A. odorata Lour. The physiological process of interaction between both hormones might have a synergistic effect on the shoot initiation and growth, cytokinin was found to proliferate embryogenesis but NAA inhibits the process (Regmi et al. 2017 in Cymbidium aliofolium). Thus, the above findings suggest that the combination of high cytokinin: auxin ratio, has proved more effective for shooting.

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In vitro rooting using different rooting hormones and growth additives from derived shoots
 
The quantity of roots and the length of the roots after four weeks of culture varied across all the treatments. The interaction between PGRs and growth additive (banana powder) showed significant results in root development (Table 3) irrespective of treatments. The root initiation was recorded earliest (31.17 days) in T3 (½ MS + 0.5 mg/L NAA + 50 g/L Banana powder). While, the maximum days taken for root initiation (43.83 days) were observed with T1. Moreover, the maximum number of roots (5.00) and length of root (2.64 cm) were also recorded in the same treatment viz., T3 and the least was recorded with T1. The findings are in line with Devi et al., (2013) who found that NAA (auxin) additions improved the rooting of A. odorata Lour. Moreover, the interaction of PGRs (auxin) with Growth additives like banana powder might have given a synergistic effect to form roots in the current study. The usage of banana powder might have helped to strengthen plantlets and develop roots. According to the variety, the chemical makeup of banana flour, banana powder, banana extract, or banana homogenate varies between 61-76.5% starch, 19-23% amylose, 2.5-3.3% protein and 0.3-0.8% lipids (Mota et al., 2000). The banana powder contains maltodextrose, which offers extra carbohydrates without having osmotic effects which boost root development. The findings were in agreement with Decruse et al. (2003) in Vanda spathulata and Alam et al. (2010) in V. teres where they concluded that a combination of auxin and banana powder obtained maximum rooting and plantlet formation. Further, Naing et al. (2011) in Coelogyne cristata also reported maximum rooting in ½ MS supplemented with auxin and Banana Powder.

Aerides odorata is one of the important and endemic orchids with great medicinal significance in Manipur. This developed in vitro immature seed culture protocol will offer a beneficial method for large-scale production of this orchid. The present study will hold significant potential for both commercial applications in the cut-flower industry and conservation endeavours aimed at restoring this orchid. Additional studies could examine the potential for scaling up this technology to produce large quantities of high-quality, disease-free planting material and to use it with more commercially significant orchid species.

The authors appreciate the facilities presented by the Central Agriculture University, Imphal, Manipur, India and are also very thankful to M/s Kwaklei and Khonggunmelei Orchids Private Limited, Imphal, Manipur for providing the un-dehisced pods of Aerides odorata var. Alba.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript. Informed consent all animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.