The rhizome of ginger is used for vegetative propagation because it is an unfertile species (Nair 2019). The rhizome is also the part of ginger used as a commercial product, so the ginger rhizomes used as ‘seed’ for cultivating in the next growing season will detract from its supply in the market. Besides, soil-borne pathogens such as bacterial wilt (Pseudomonas solanacearum), rhizome rot disease (Pythium myriotylum, Pythium spinosum, and Pythium sylvaticum), and nematodes (Meloidogyne spp.) are easily transmitted during vegetative reproduction, carried by the ginger rhizomes (Kasilingam et al. 2018; Abed et al.2020). We all know that in-vitro propagation not only could be a suitable alternative for the effective production of ginger seedlings, but also can eliminate the transmission of pathogens through the ginger rhizomes. Therefore, ginger tissue culture seedlings not only solve the problem of using a large proportion of marketable ginger as seed, but also ensures disease-free of ginger seeds. At the same time, the asexual propagation ginger for producing germplasm for breeding purposes has proven difficult, but we can use tissue culture technology to mutate ginger in vitro, so as to produce new germplasm (Jagadev et al. 2008; Mohanty et al. 2008; Zhou et al. 2020). However, the cost of transplanting cultured seedlings to the field accounts for 40–60% of the total costs of the tissue culture seedling production process. Therefore, improving the survival rates of ginger tissue culture seedlings during the transplantation stage and reducing production costs are urgently needed so that tissue culture technology can be applied to commercial production.
For transplantation, the in-vitro grown ginger plantlets were thoroughly washed with tap water to remove residual agar from roots. Then they were immersed in 0.2% aqueous Bavistin solution (fungicide) for 15–20 min and washed with tap water. The treated plantlets were then transplanted to pots filled with the different substrates and cultured in a greenhouse (Mohamed et al. 2011). Mohamed et al. (2011) found that the acclimatized rooting ginger tissue culture seedlings could be domesticated in peatmoss + sand + vermiculite with a survival rate of 60%. The regenerated ginger plantlets were then planted in a potting mixture of equal proportions garden soil, sand, and vermiculite and had an 85% survival rate (Samsudeen et al. 2000). Hung et al. (2018) found that when Dendrobium officinale could be acclimatized in plastic pots containing a mixture of 1:1 humus soil and coconut bran, the survival rate was about 31%. However, in this study, the rootless ginger tissue culture seedlings in seedling tray were incubated in a growth chamber and successfully acclimatized with 100% survival.
Among the six substrates selected in this study, the vermiculite + peat (1:1(v/v)) substrate was the most suitable. This may have been because vermiculite facilitates good air permeability and water absorption, while peat is rich in organic matter and humic acids. Although there were many substrates suitable for the cultivation of ginger tissue culture seedlings, such as peat + vermiculite + sediment (1:1:1(v/v/v)) (Qiu et al. 2020), in-vitro rooted ‘Bentong’ ginger plantlets were acclimatized in a growing media mixed of soil + coco peat + vermiculite (1:1:1(v/v/v)) (Zahid et al. 2021). The compositions of the soil and sediment were not determined, but the peat and vermiculite were known. Therefore, the substrate formulation of this study is easier to commercialize.
This obtained result was in line with the findings of Jagadev et al. (2008), who observed that for rooting of Z. officinale Rosc, MS supplemented with NAA (0.5 mg/L) was more effective and resulted in the maximum number of roots per shoot. Different nutritional liquids also affected the rooting rate. There have been many reports in this regard, such as Rout et al. (2008) who indicated that excised shoots were rooted on half-strength MS basal salts supplemented with 0.25 mg/l IBA or IAA and 20 g/l (w/v) in Acacia chundra. Kambaska et al. (2009) concluded that in-vitro shoots of Z. officinale Rosc rooted best when half strength MS basal medium supplemented with 2.0 mg/L NAA was used. Mohamed et al. (2011) found that shootlets became highly rooted when half strength B5 medium was supplemented with 1.0 mg/L NAA. In-vitro root induction in ginger is further enhanced by supplementing the culture medium with different nutrients (Abbas et al. 2011; Mehaboob et al. 2019). Determining the optimum type and concentration of nutrition can significantly enhance the in-vitro root induction of ginger and facilitate the acclimatization and successful establishment of the in-vitro plantlets in field conditions (Mehaboob et al. 2019; Jualang et al. 2015). In this study, the plant height and root number were optimized when rootless ginger tissue culture seedlings were planted with MS + 0.5 mg/L NAA.
Loose and breathable substrate combined with appropriate humidity is necessary for rooting in the process of photoautotrophic micropropagation. Too much water and low oxygen content in the substrate are not conducive to the respiration of the plant and can easily cause base rot and reduce the rooting rate. However, low water contents in the substrate can make it difficult for plants to obtain enough water from the substrate, resulting in plant water loss, which can affect metabolism and result in a low rooting rate. Proper humidity can keep the plant moist, provide water for metabolism, and ensure the supply of oxygen to a certain extent. This is an important measure for maximizing the rooting rate. The goal of this study was to seek the optimum relative humidity (RH) levels for ginger photoautotrophic micropropagation. The results indicated that a moderate RH level of 80% was best because it had the best rooting effect for rootless tissue culture seedlings. The plant height and stem diameter of rootless ginger tissue culture seedlings were lowest in the 60% RH treatment, and the plant height, stem diameter, and root weight were relatively low in the 90% RH treatment. Previous studies have reported that a moderate level of water stress may actually be necessary to initiate root formation and optimize rooting success (Lebude et al. 2004, Tombesi et al. 2015). This study supports the previous reports that intermediate RH levels may actually promote root formation (Tyler et al. 2017)
Microbial contamination of plants can adversely affect plant tissue culture. These microbes can compete for nutrients, increase culture mortality, and may result in variable growth, tissue necrosis, and reduced shoot proliferation and rooting (Afolabi, 2009). In this study, rootless tissue culture seedlings of ginger were planted in vermiculite and peat supporting material, which were sugar free and had almost no bacterial or fungal contamination. However, because of the semi-enclosed and high humidity environment, some microbial growth did occur. Adding 0.1% Metalaxyl-M·Hymexazol to the substrate during the photoautotrophic micropropagation of ginger effectively inhibited the growth of bacteria. Furthermore, a certain concentration of Metalaxyl-M·Hymexazol not only had an antibacterial effect, but also promoted the rooting of ginger seedlings.
Micropropagation is an advanced technology capable of producing a large number of genetically superior and pathogen-free plants rapidly and in a small amount of space. However, the widespread application of micropropagation is still limited by high production costs, which are mostly attributed to the significant loss of plants grown in vitro due to microbial contamination, poor rooting, and low survival rates during the ex-vitro acclimatization stage (Kozai et al. 2001). Labor costs for rooting and acclimatization of plantlets account for approximately 60% of the total production costs in conventional micropropagation. Furthermore, there is relatively high mortality in plantlets due to the extreme environmental stresses experienced during the acclimatization stage. In-vitro grown explants and plantlets have been considered to have relatively low photosynthetic ability and to require sugar as a carbon and energy source for their heterotrophic or mixotrophic growth. Therefore, it is important that studies focus on optimizing the nutrient medium in vitro and mixotrophic micropropagation for specific plants and on determining when and how the medium should be applied to explants/plantlets in vitro. In this study, photoautotrophic micropropagation (PAM) of ginger was optimized using vermiculite + peat (1:1(v/v)) as substrate combined with MS + 0.5 mg/L NAA + 0.1% Metalaxyl-M·Hymexazol + 80% RH. After 40 days of culture, ginger seedlings grown in vitro were successfully bred with a rooting rate of 100.0%.