Field Performance of Disease-Free Plants of Ginger Produced by Tissue Culture and Agronomic, Cytological, and Molecular Characterization of the Morphological Variants

Abstract

Ginger (Zingiber officinale Rosc.) is an important spice crop valued for its flavored and medical properties. It is susceptible to soil-borne diseases, which can cause considerable economic loss to growers. In vitro culture is feasible for the propagation of disease-free ginger plants, but has several disadvantages when producing seed rhizomes that can be commercially used, such as long cultivation cycles (usually 2–3 years) and occurrence of somaclonal variation. In this study, dynamic changes in the morphological characteristics of in vitro-propagated disease-free plants of ‘Wuling’ ginger were evaluated by continuous observation and measurement at 30-day intervals, and morphological variants were screened and characterized by agronomic, cytological, and molecular analysis at harvest. Results showed that the plants grew rapidly within 120 days after planting, and the most active growth period was from 60 to 120 days. Eight plants with clear and stable morphological differences were screened out from approximately 2000 plants grown in the field, and they could be classified into two groups (VT1 and VT2) based on tiller number, plant height, leaf color, and leaf shape. By flow cytometry analysis and chromosome counting, the VT1 was confirmed to be diploid, with the shortest plant height, the largest number of tillers and rhizome knobs, and the smallest tiller diameter and rhizome size among the three types of plants. The VT2 was mixoploid, consisting of diploid and tetraploid cells, with significantly reduced tiller number and rhizome knobs, significantly larger stomatal guard cells/apertures, and significantly lower stomatal density. SSR analysis detected DNA band profile changes in six out of the eight variants, including one plant of the VT1 and all the VT2 plants. The findings of this study might contribute to the commercial production of disease-free seed rhizomes in ginger, and the characterized somaclonal variants could provide useful germplasm resources for future breeding.

In Vitro Microrhizome Production, Genetic Homogeneity Assessment, and Field Performance Evaluation in Ginger

“…Although the most suitable medium for microrhizome induction (A3B3C1) contains a high amount of BAP (3.0 mg•L −1 ) and sucrose (100 g•L −1 ), the genetic homogeneity of the TC, MR, and FMR plants in this study was confirmed by flow cytometry analysis and SSR characterization. Genetic instability and somaclonal variation have rarely been reported in ginger [9,48], possibly due to the high genetic stability during in vitro culture [9]. However, the limited use of five plants and ten pairs of SSR primers in this study may provide insufficient genetic information of the regenerated population.…”

“…Plants of disease-free Zingiber officinale Rosc. cv Fengtou were previously obtained [9]. The plants were maintained on Murashige and Skoog (MS) basal medium [37] supplemented with 1 mg•L −1 6-benzylaminopurine (BAP), 0.2 mg•L −1 indole butyric acid (IBA), 30 g•L −1 sucrose, and 7.5 g•L −1 agar (pH 5.8), with a subculture interval of 60 days.…”

“…DNA concentration and purity were determined on a NanoDrop One C spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). For simple sequence repeat (SSR) amplifications, ten randomly selected gingerspecific microsatellite loci (Table S1) retrieved from Zhao et al [9] were analyzed to assess the genetic homogeneity. The PCR reaction was performed in a total volume of 20 µL containing 2 µL of 25 ng•µL −1 temple DNA, 1.0 µL of each forward and reverse primer (10 µM), 10 µL of 3G Master Mix for polyacrylamide gel electrophoresis (Vazyme, Nanjing, China), and 6.0 µL ddH 2 O.…”

“…Therefore, it is imperative to cultivate pathogen-free seedling rhizomes via a cost-effective method with a high multiplication rate to ensure success in cultivating this crop species. In vitro culture has been extensively used to produce disease-free plants in numerous crop species [6,7], including ginger [8,9]. However, ginger plants regenerated in vitro generally produce low yields and small rhizomes that are not suitable for commercial use in the first growing season [10].…”

“…However, ginger plants regenerated in vitro generally produce low yields and small rhizomes that are not suitable for commercial use in the first growing season [10]. Typically, it Agronomy 2024, 14, 747 2 of 18 takes 2-3 growing cycles to produce a yield and seed rhizome size that is acceptable to the grower [9,10]. Moreover, conventional tissue culture plants require several time-consuming and labor-intensive procedures before plant transplantation, such as removing residual media and acclimating the plants [11,12].…”