The rhizome of ginger is used for vegetative propagation because it is an unfertile species with a low reproduction coefficient (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 rhizomes fragments (Kasilingam et al. 2018; Abed et al.2016). Therefore, in-vitro propagation using tissue culture could be a suitable alternative for the effective production of ginger and ginger seedlings that can eliminate the transmission of pathogens. This will reduce costs as the bulkiness of ginger rhizomes as planting material makes their handling costly and laborious. Furthermore, to improve asexual crop species, most efforts have been restricted to evaluating individuals and selecting for desired traits. However, ginger crops may be improved by clonal multiplication through the induction of multiple shoots, as has been reported by several studies (Jagadev et al. 2008; Mohanty et al. 2008). 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 this rapid propagation 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 in 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 were domesticated in the peatmoss + sand + vermiculite treatment, with a development 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 was 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 in-vitro rooted ginger plantlets in culture vessels 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).
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. The rooting effect was different when different nutrient solutions and auxin were added to the substrate. 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 auxins (Abbas et al. 2011; Mehaboob et al. 2019). Different types and concentrations of auxins have varying effects on in-vitro root induction in different Zingiberaceae species (Mehaboob et al. 2019; Jualang et al. 2015). Determining the optimum type and concentration of auxin 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. 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.
This study also sought to identify optimal relative humidity (RH) range 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. There were no significant differences in ginger seedling growth among different humidity conditions. Generally, high RH levels decrease transpiration and water loss, which supports the high turgor pressure necessary for cell expansion and growth in adventitious roots and prevents desiccation (Loach 1988). This may have been why the plant height and stem diameter of rootless ginger tissue culture seedlings were lowest in the 60% RH treatment. However, our results showed that 80% RH was adequate for rapid rooting, whereas higher levels of RH reduced the rooting success. 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), and lower water potentials actually had the highest rooting and lowest mortality (Tombesi et al. 2015). One ecological explanation could be that plants put resources into growing structures that enable them to obtain more resources when stressed. Under water stress, ginger tissue culture seedlings tended to invest more resources in root growth.
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). In conventional photomixotrophic micropropagation, the sugar-containing medium must be carefully washed off the plant before transplanting to ex-vitro conditions. In this study, rootless tissue culture seedlings were planted in vermiculite and peat supporting material, which were sugar free and had with almost no bacterial and fungal pollution. However, because of the semi-enclosed and high humidity environment, sometimes there was some microbial pollution. 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.
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 hetero 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%.