Stretching approximately 4,880 kilometers, the Mekong River ranks as the world's tenth longest river. Originating in China's Himalayan region, the river courses through several countries—the Republic of the Union of Myanmar, Laos, Thailand, and Cambodia, and finally empties into the South China Sea in Vietnam. In Thailand alone, the Mekong River spans 980 kilometers. Serving as a pivotal cornerstone of civilization, the river is a fertile and ecologically diverse waterway. It hosts remarkable biodiversity, including over 1,100 fish species, in excess of 430 mammalian species, and a plethora of reptiles, amphibians, and birds, totaling over 800 species. Additionally, the river's ecosystem is rich in plant life, harboring more than 20,000 plant species and over 1,200 unique species (Commission, 2010).
Cryptocoryne, belonging to the Araceae family, is an aquatic plant that is native to the riparian zones of the Mekong River. Characterized by its vivid coloration and distinctive leaf shapes, certain Cryptocoryne species have gained recognition as ornamental aquatic plants, leading to their commercialization. Particularly in the upper northeastern areas of the Mekong River in Thailand, a diverse array of Cryptocoryne species can be found. These consist of Cryptocoryne crispatula var. yunnanensis, C. crispatula var. crispatula, C. crispatula var. tokinensis, C. loeiensis, and their hybrids (Bastmijer et al., 2010). Predominantly located in sand and bedrock areas like the Chiang Khan District, Pak Chom in Loei Province, and Pan Khot Saen Krai in Nong Khai Province, these varieties thrive in specific ecological niches. These plants flourish and bloom from December to April during the dry season, displaying a spectrum of floral characteristics, such as varying tube lengths, spiral lengths, and spathe limb colors. During the flood season from May to October, they enter a dormant phase. However, the operation of hydropower dams upstream of Loei Province has induced significant diurnal water level fluctuations during the dry season (Lu & Chua, 2021; Raviwan & Konyai, 2012). These altered hydrological conditions, which have been observed recently, negatively influence the growth and reproductive patterns of the Cryptocoryne species. This environmental disruption poses a threat to their survival, with a particular impact on C. crispatula var. yunnanensis, which can be distinguished by its short flower tube and is currently listed as an endangered species in China (Bastmijer et al., 2010; Qin et al., 2017). Such ecological changes raise concerns about the potential decline or even extinction of these species in the foreseeable future.
Plant tissue culture technology is a vital tool for the restoration of various plant species. This method utilizes explants, such as shoot tips, lateral buds, and leaves. The process involves sterilizing these explants and then cultivating them in an artificial medium that has been enriched with plant growth regulators (PGRs). In tissue culture biotechnology, the investigation of the diverse regenerative pathways that is facilitated by PGRs is crucial. Cytokinins, including Benzyladenine (BA), Kinetin, and Thidiazuron (TDZ), form a primary class of PGRs. These phytohormones play essential roles in numerous plant growth functions. Their scope of influence extends to cellular division, photosynthesis, chloroplast differentiation, regulation of leaf senescence, and nutrient metabolism. Importantly, they are vital in maintaining the functionality of the meristem, especially in the shoot and root systems (Anbazhakan et al., 2022Asghar et al., 2023; Li et al., 2021). Auxin, another pivotal phytohormone, has been extensively researched for its role in plant development. In this context, Naphthaleneacetic Acid (NAA) is recognized as a crucial inducer of somatic embryos. The impact of NAA, especially when mediated by auxin-inducing promoters, is particularly significant in the early stages of embryonic development. This technology leverages the principle of totipotency, which enables the generation of complete plants via organogenesis or somatic embryogenesis (Asghar et al., 2023, Choudhary et al., 2020). Specific protocols are employed for different plant species. For instance, in the case of Cryptocoryne, a disinfection process using NaOCl and mercuric chloride at varying concentrations and durations has been established (Klaocheed et al., 2020; Stanly et al., 2011). The successful cultivation of Cryptocoryne wendtii has been achieved by using Murashige and Skoog (MS) medium, which has been supplemented with 3–4 mg/L BAP and 1 mg/L NAA or IBA. For this species, this composition has been shown to promote effective shoot and root development (Klaocheed et al., 2020; Unal et al., 2019).
Somaclonal variation in the process of in vitro plant propagation is influenced by numerous factors, including the type of tissue being used, the source of the explants, the concentrations and types of plant growth regulators, and the duration of the culture cycle, which can accelerate the induction of mitotic cycles within a limited time period (Noceda & Steinmacher, 2020; Zeid et al., 2022). Given this potential for variation, assessing the genetic stability of micropropagated plants is crucial in order to ensure their fidelity to the parent plant. To identify and confirm any genetic abnormalities, various techniques, such as isozymes analysis, cytological examinations, and molecular markers, are employed. Among these, Inter Simple Sequence Repeat (ISSR) analysis has been widely utilized for verifying the genetic uniformity and integrity of both propagated and parent plants. ISSRs consist of DNA fragments ranging from 100 to 3000 base pairs, which are located between adjacent, inversely oriented microsatellite regions. These sequences are amplified based on polymerase chain reactions with 16–18 base pairs single primers derived from microsatellite core sequences, as well as based on a few selective nucleotides extending into adjacent non-repeat regions (Godwin et al., 1997). The utility of ISSR markers in establishing genetic homogeneity has been demonstrated in micropropagated plants of various species, including Crinum malabaricum, Reseda pentagyna, and Teucrium polium (Al-Qurainy et al., 2018; Priyadharshini et al., 2020; Saadat et al., 2023). This underscores the importance of ISSR markers as reliable tools that can be utilized for genetic assessment in the field of plant tissue culture and propagation.
This study centered on the optimization of in vitro surface disinfection procedures using Cryptocoryne crispatula var. yunnanensis. The research aimed at evaluating the effects that various cytokinin and auxin concentrations could have on the in vitro shoot regeneration and proliferation. Additionally, the study investigated the ex vitro root regeneration and the acclimatization process. An assessment of the genetic integrity of the newly propagated offspring was conducted using ISSR markers. It was expected that this approach would offer crucial insights into the effective use of plant growth regulators, not only to enhance in vitro shoot formation, but also to promote ex vitro root development, while also ensuring the genetic stability of the propagated plants.