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ASHS 2024 Annual Conference

 

Mutation Breeding of Salvia coccinea with Ethyl Methanesulfonate

Authors:
Rebekah C.I. Maynard Department of Horticulture, University of Georgia, Athens, GA 30602, USA

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John M. Ruter Department of Horticulture, University of Georgia, Athens, GA 30602, USA; and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA 30602, USA

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Abstract

Salvia coccinea is a valuable flowering annual that attracts hummingbirds and bees to the garden, but few cultivars are commercially available. There is a limited range of petal colors and no leaf variegation. This research aimed to improve the ornamental value of S. coccinea by inducing mutations with ethyl methanesulfonate (EMS). The standard, red-flowered species was selected for treatment by exposing seeds to 0%, 0.4%, 0.8%, or 1.2% EMS for 8, 12, or 24 hours. The optimal treatment rate was determined to be 1.2% EMS for 8 hours, which generated desirable mutations near the median lethal dose (LD50). The M1 population had a 53% germination rate and was completely morphologically uniform. By the M2, mutations included differences in leaf shape and flower size in addition to albina, chlorina, virescens, and chimeral chlorophyll changes. A 1% mutation rate was achieved in this breeding program with seven unstable mutations and six stable mutations. The normalized difference vegetation index (NDVI) values were measured to determine differences in chlorophyll content between lethal albina mutations, chartreuse chlorina and virescens mutations, and typical leaf color. Future work will investigate the stability and heritability of chlorophyll variegation by hybridizing these selections with coral-flowered accessions of S. coccinea.

Salvia coccinea is an attractive flowering plant used as a self-seeding annual or herbaceous perennial in the landscape. On average, it grows to 1 m in height with uniform branching, pubescent green leaves, and red to white flowers (Clebsch 2003). S. coccinea is a vital pollinator plant for hummingbirds and has naturalized in the southeastern United States. Its native range is uncertain but is believed to extend from Mexico to Central America or Brazil (Wester and Claßen-Bockhoff 2011). Compact cultivars have been selected such as Lady in Red with crimson flowers and Summer Jewel Pink with bicolored white and pink petals. However, limited breeding work has been done to improve the species further.

There is a continual market demand for ornamental plants with new characteristics. If limited natural variation exists, mutation breeding can improve a crop by artificially inducing genetic variation. This breeding technique has been shown to cause phenotypic variations in color, flower shape, plant height, and leaf chimeras (Datta and da Silva 2006). Chlorophyll mutations are the most common and reliable way to determine the efficacy of treatments (Patial et al. 2017); however, most induced mutations are recessive and cannot be segregated until the M2 generation (Toker et al. 2007).

Although unique traits can be isolated using mutation breeding, the occurrences are random, and induction treatments can reduce germination and subsequent growth of seedlings. Therefore, large populations must be treated to increase the chances of achieving a plant with desirable characteristics (Toker et al. 2007). Nine categories and sub-categories of chlorophyll mutations were described by Gustafsson (1940). These categories can be used to distinguish a mutated population based on leaf coloration and pattern. Unfortunately, because of a disruption in chlorophyll production, many mutants are lethal. However, observing chlorophyll mutations helps determine an appropriate mutagen concentration to maximize induced genetic variation (Singh et al. 2019).

The color differences caused by chlorophyll mutations can be described numerically using spectrophotometer readings. Chlorophyll absorbs solar radiation and fluoresces in the red (685–690 nm) and far-red (730–740 nm) regions of the electromagnetic spectrum. Therefore, chlorophyll content, and subsequently leaf greenness, can be determined by comparing the ratio of red and far-red reflectance (Buschmann 2007). The NDVI describes relative amounts of red and far-red light reflected from leaves. Leaves with low chlorophyll content reflect more red light and lower the NDVI value (Glenn and Tabb 2019). Therefore, NDVI readings can be used to assess leaf greenness and photochemical activity.

Mutations can be induced through radiation or exposure to a chemical mutagen (Datta and da Silva 2006). In a mutation study conducted on Arabidopsis thaliana, 11 different physical and chemical mutagens were tested to determine which had the most significant effect. The chemical mutagen EMS was shown to have the highest mutation and survival rate compared with the other treatments (McKelvie 1963). In similar studies, EMS was found to generate the most significant mutations in Abelmoschus esculentus (Gupta et al. 2017), Delphinium malabaricum (Kolar et al. 2015), and Vigna umbellata (Patial et al. 2017) compared with other chemical and physical mutagens.

The LD50 is commonly used to generate many mutated plants without significantly reducing the population from the toxic effects of the mutagen (Yadav et al. 2016); however, prolonged exposure to EMS may have deleterious effects on phenotypic characteristics without reducing seed germination. For example, in research conducted by Jiang et al. (2014), an increase in EMS concentration and exposure time was determined to reduce flower production in Petunia without reducing seed germination (Jiang et al. 2014). Furthermore, Khalatkar (1976) studied the effect of EMS uptake in dry Hordeum vulgare seeds compared with seeds presoaked in water. It was found that dry seeds did not develop with chlorophyll chimeras, but presoaking resulted in a 9% chlorophyll mutation rate without a significant decrease in germination. Therefore, it was recommended to presoak seeds before treatment with EMS to increase mutation frequency (Khalatkar 1976).

EMS mutagenesis has been used to improve the appearance of several ornamental plants. These improvements included the introduction of golden and white foliage in Delphinium (Kolar et al. 2015), novel and stable petal colors in Dendranthema (Latado et al. 2004), increased flowering ability in Gladiolus (Bahajantri and Patil 2013), and unique petal fringe in Saintpaulia (Fang and Traore 2011). Although this breeding technique is widely used, it has not been well documented for Salvia. This study aimed to induce genetic variation in S. coccinea by exposure to EMS and isolate plants with improved phenotypic characteristics for use in the landscape.

Materials and Methods

Expt. 1.

An experiment was designed to determine the optimum treatment parameters to induce mutations in S. coccinea. Twelve different treatment groups were tested by varying exposure time and concentration of the chemical mutagen EMS to form a two-way factorial. The first factor, with three levels, tested the mutagenic impact of exposure time by using an 8-, 12-, or 24-h treatment period. The second factor, with four levels, tested the impact of mutagen concentration by using 0%, 0.4%, 0.8%, or 1.2% EMS. Finally, the experiment had three replicates of 18 seeds in each treatment group.

Mature seeds were harvested from a stock plant maintained under greenhouse conditions in January 2020. Seeds were presoaked in deionized water for 12 h to increase their ability to imbibe EMS solution. For the control group, a beaker was prepared with only deionized (DI) water. EMS was diluted with DI water to form the appropriate solution concentrations for treatment groups. Once the solutions were created, seeds were randomly assigned into their treatment groups. The beakers were covered with parafilm and continuously agitated on a mechanical shaker for 8, 12, or 24 h at 200 rpm (New Brunswick Scientific, Edison, NJ, USA). After the treatment period, the beakers were removed from the shaker, and their solution was decanted off. Seeds were rinsed for 5 seconds with DI water and decanted three times to remove residual EMS.

The treated seeds were individually sown on PRO-MIX HP substrate with biofungicide and mycorrhizae (Premier Tech Horticulture, Quakertown, PA, USA) in a 128-cell (45 mL) plug flat. Each flat contained four treatment groups. The flats were arranged in a randomized complete block design across three benches in a greenhouse at the University of Georgia Horticulture Research Farm in Athens, GA, USA (33.8870, −83.4201). The flats were grown under 70% shade with no supplemental lighting and irrigated from below. Daytime temperatures were set to 24 °C, and nighttime temperatures were set to 19 °C.

Cotyledon emergence was recorded through daily observation. Once the seedlings had two sets of true leaves, they were moved up into 1.05-L square pots with PRO-MIX HP substrate, fertilized with 3.5 g of 8- to 9-month Osmocote Plus 15–9–12 (15N–4.4P–10.0K) (ICL Specialty Fertilizers, Summerville, SC, USA) and moved into full sun. The significance of EMS concentration and exposure time effects on seed germination was tested with a two-way analysis of variance using the programming language R (RStudio, Boston, MA, USA). The M1 population was self-pollinated, and 60 seeds were collected from each treatment group to form the M2 generation. Mutations were described by their leaf color, leaf shape, and floral structures.

Expt. 2.

Based on the LD50 of the M1 population and the observed mutations in the M2 population from the first experiment, 1.2% EMS for 8 h was selected as the optimum treatment for inducing mutations in S. coccinea. Therefore, this treatment concentration and exposure time were used to treat a large population of seeds. In January 2021, seeds were harvested from an S. coccinea greenhouse stock plant and randomly assigned into treatment groups. The treated population consisted of six replicates of 100 seeds, and the control had six replicates of 20 seeds. All seeds were presoaked and treated with EMS as previously described. After the treatment period, seeds were rinsed three times with DI water and sown individually in 200-cell (22 mL) plug flats on PRO-MIX HP substrate. Each flat had one treatment group and one control and was left under a shaded bench with the same greenhouse conditions as the first part of the experiment.

A 53% germination rate was observed in the M1 population. Once the surviving seedlings had two sets of true leaves, they were moved up into 1.05-L square pots, fertilized with 3.5 g of 8- to 9-month Osmocote Plus 15–9–12 and moved into full sun. The plants were self-pollinated, and four seeds were collected from each plant to form the M2 population. These seeds were sown individually in 200-cell (22 mL) plug flats on PRO-MIX HP substrate. They were left to germinate on a shaded bench and subjected to the same greenhouse conditions from the first experiment. Mutations in the M2 population were described using the same parameters as the first experiment. A handheld UV-Vis spectrophotometer (PolyPen RP 410 UVIS; Photon Systems Instruments, Drásov, Czech Republic) was used to measure the spectral reflectance of leaves with uniform chlorophyll mutations and obtain the NDVI. Five representative leaves of each mutation were averaged to calculated mean and standard deviation NDVI values.

Results and Discussion

Expt. 1.

In the M1 population, a statistically significant difference was found in germination for both EMS concentration [f(3) = 141.59, P < 0.001] and exposure time [f(2) = 101.50, P < 0.001]. In addition, a significant interaction was found between the two factors at higher concentrations of EMS [f(6) = 37.87, P < 0.001]. Figure 1 shows a significant decline in cotyledon emergence with increasing EMS concentration and exposure time. A decline in seedling germination with increasing EMS concentration has been well documented with many other species, including Abelmoschus esculentus (Baghery et al. 2016), Oryza sativa (Talebi et al. 2012), and Sarcococca confusa (Hoskins and Contreras 2019).

Fig. 1.
Fig. 1.

Germination percentage was measured after treating a population of Salvia coccinea seeds with 0%, 0.4%, 0.8%, or 1.2% ethyl methanesulfonate (EMS) for 8, 12, or 24 h. Cotyledon emergence declined significantly with higher levels of EMS and longer exposure times.

Citation: HortScience 58, 5; 10.21273/HORTSCI17092-23

In this study, all plants in the M1 were morphologically uniform. This result was expected because most mutations are recessive (Toker et al. 2007). The mutation effect of EMS on S. coccinea was observed in the M2 generation chlorophyll mutations, leaf deformation, and changes in floral structures. The leaf chlorophyll mutations were described using classification by Gustafsson (1940). These mutations included albina, chlorina, virescens, and other nonuniform chlorophyll mutations not described by Gustafsson (1940). The albina mutation is characterized by a lack of chlorophyll and carotenoids resulting in completely white leaves. In chlorina mutants, leaves develop with a uniform, stable chartreuse color. Virescens mutants are similar to chlorina mutants at the early developmental stages; however, leaf color returns to normal in the mature plant (Gustafsson 1940).

Two stable mutated plants were observed in the M2 population from separate treatment groups. These mutations are shown in Fig. 2B and C. The first mutated plant was observed in the group treated with 1.2% EMS for 8 h. This plant had a visible chlorina mutation, the overall growth habit was significantly more compact, and both the leaves and flowers were smaller than the species. The leaf shape became more ovate than the parent but maintained serrated leaf margins and typical reproductive structures. Reduction in growth has also been observed in a yellow-green leaf mutant of Betula. This reduced growth was believed to be the result of lower chlorophyll production (Ren et al. 2018).

Fig. 2.
Fig. 2.

Stable mutations observed in the M2 population of Salvia coccinea treated with ethyl methanesulfonate (EMS). (A) S. coccinea untreated control, (B) leaf deformation from Expt. 1 with exposure to 0.8% EMS for 12 h, and (C) chlorina mutation from Expt. 1 with exposure to 1.2% EMS for 8 h. (D–G) Mutations from Expt. 2 with exposure to 1.2% EMS for 8 h. (D) Sectorial chimera, (E and F) differential gene expression, and (G) leaf deformation.

Citation: HortScience 58, 5; 10.21273/HORTSCI17092-23

The second mutated plant was found in the M2 population from seeds exposed to 0.8% EMS for 12 h. No chlorophyll mutations were observed in this plant; however, the leaves were densely covered with fine hairs, giving a hazy blue-gray appearance. This mutation displayed significantly deformed leaves with revolute leaf margins. The flowers lacked anthers and had reduced floral lobes. Changes in leaf shape and male sterility were also observed in EMS mutants of Arabidopsis (McKelvie 1963). No chlorophyll mutations occurred below 0.8% EMS in this experiment. Although mutations were observed at 1.2% EMS for 8 h and 0.8% EMS for 12 h, no mutations were observed at longer exposure times for either of these concentrations.

Expt. 2.

In the second study, mutations were observable at the seedling stage of the M2. The greatest number of chlorophyll mutations in this population were unstable virescens mutations (Fig. 3B–D). The leaves initially were chartreuse but later matured to the typical leaf color associated with S. coccinea. Virescens mutations were also the most commonly occurring chlorophyll mutation in Vigna umbellata (Gustafsson 1940; Patial et al. 2017). Two lethal albina mutants (Fig. 3E and F) had no production of chlorophyll or carotenoids. These did not survive beyond two sets of true leaves. Albina mutants have been described as entirely lethal in other species, such as Cajanus cajan (Etther et al. 2019), Capsicum annum (Kumar et al. 2000), and Vigna umbellata (Patial et al. 2017).

Fig. 3.
Fig. 3.

Unstable mutations of Salvia coccinea observed in Expt. 2 from exposure to 1.2% ethyl methanesulfonate for 8 h. (A) S. coccinea untreated control, (BD) virescens chlorophyll mutations, (E and F) lethal albina chlorophyll mutations, and (G and H) leaf deformations.

Citation: HortScience 58, 5; 10.21273/HORTSCI17092-23

The lethal nature of albina mutants can be explained by their lack of chlorophyll. Photochemical reflectance measurements are commonly used to determine variations in vegetation and subsequent photosynthetic activity (Chen et al. 2014; Chu et al. 2019; Gamon et al. 2015). This study compared the chartreuse leaves observed in the viridis and virescens mutants with white leaves of the albina mutant and normal S. coccinea leaves by measuring their NDVI. The difference in leaf color is shown in Fig. 4. The NDVI values of the green, chartreuse, and white leaves were 0.722 ± 0.009, 0.442 ± 0.003, and 0.016 ± 0.002, respectively. Lower NDVI values correspond with lower chlorophyll content, as observed in the less pigmented leaves (Glenn and Tabb 2019). These NDVI readings explain the difference in pigmentation among the mutations and the inability of the albina mutant to survive past the seedling stage with reduced photosynthetic activity.

Fig. 4.
Fig. 4.

Mutants from the M2 population of Salvia coccinea treated with 1.2% ethyl methanesulfonate for 8 h. Pictured on the left is an albina chlorophyll mutation, the center leaf is a chlorina mutation, and the leaf on the right is chlorophyll expression in an untreated Salvia coccinea control.

Citation: HortScience 58, 5; 10.21273/HORTSCI17092-23

Three other nonuniform but stable chlorophyll mutations were isolated from this population, which Gustafsson’s (1940) classifications did not describe. As shown in Fig. 2D, one plant appeared to be a sectorial chimera with half of an entire leaf lacking chlorophyll. Sectorial chimeras form when a mutation in the chloroplast DNA creates blocks of distinct tissue in all three apical layers (Marcotrigiano 1997). The other two stable, nonuniform mutations (Fig. 2E and F) had splotches of light green and white tissue dispersed across the leaf. Because these mutations did not show a pattern relating to the apical cell layers, they were likely caused by a mutation in the chloroplast. Cells with both mutated and normal chloroplasts are known as heteroplastic cells. As the cell divides, the chloroplasts are sorted into cells containing either mutated or normal DNA, resulting in a mosaic appearance (Marcotrigiano 1997).

In addition to chlorophyll mutations, three plants with leaf deformations were observed in the M2. One leaf mutation was stable and resulted in larger, more cordate leaves compared with the untreated species, as shown in Fig. 2G. The other two mutations were unstable leaf deformations. The altered leaves were lanceolate with entire leaf margins (Fig. 3G and H). Eventually, the mature plants reverted to typically shaped leaves. Changes in leaf shape from EMS exposure have been described in Abelmoschus esculentus (Gupta and Sood 2019), Chrysanthemum morifolium (Nasri et al. 2021), and Cucumis sativus (Chen et al. 2018).

Six stable mutants were isolated from the M2 populations of both experiments and are summarized in Table 1. This research may be continued by self-pollinating and seed propagating plants from the M2 to look for mutations in later generations. In Chrysanthemum indicum, mutations from EMS exposure were not isolated until the M3 (Purente et al. 2020). Future work could look at mutation frequency through the M3 to check for mutations in the later generations.

Table 1.

Salvia coccinea was induced to form mutations with ethyl methanesulfonate (EMS). The table is a summary of the stable mutations from Fig. 2 isolated in the M2 population. (A) S. coccinea untreated control, (B) leaf deformation from Expt. 1 with exposure to 0.8% EMS for 12 h, and (C) chlorina mutation from Expt. 1 with exposure to 1.2% EMS for 8 h. (D–G) Mutations from Expt. 2 with exposure to 1.2% EMS for 8 h. (D) Sectorial chimera, (E and F) differential gene expression, and (G) leaf deformation.

Table 1.

These mutant populations may be further improved by testing the heritability and stability of selected mutants. Early studies in maize indicated that chlorophyll is inherited maternally (Anderson 1923). In a more recent study, however, maternal inheritance was not detected in mutant Arachis hypogea. Chlorophyll mutants reciprocally crossed with a normal green-leaved accession produced mutants that were recessive but heritable. In the F2 population, progeny segregated in a 3:1 ratio of normal to albino-virescent leaves (Branch and Brown 2019). S. coccinea mutants from this study may be further improved through reciprocal crosses to accessions with different flower colors. Mutants were autogamous, indicating they were male and female fertile. If chlorophyll mutations are heritable, improved lines may be selected for leaf variegation in multiple flower colors.

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  • Fig. 1.

    Germination percentage was measured after treating a population of Salvia coccinea seeds with 0%, 0.4%, 0.8%, or 1.2% ethyl methanesulfonate (EMS) for 8, 12, or 24 h. Cotyledon emergence declined significantly with higher levels of EMS and longer exposure times.

  • Fig. 2.

    Stable mutations observed in the M2 population of Salvia coccinea treated with ethyl methanesulfonate (EMS). (A) S. coccinea untreated control, (B) leaf deformation from Expt. 1 with exposure to 0.8% EMS for 12 h, and (C) chlorina mutation from Expt. 1 with exposure to 1.2% EMS for 8 h. (D–G) Mutations from Expt. 2 with exposure to 1.2% EMS for 8 h. (D) Sectorial chimera, (E and F) differential gene expression, and (G) leaf deformation.

  • Fig. 3.

    Unstable mutations of Salvia coccinea observed in Expt. 2 from exposure to 1.2% ethyl methanesulfonate for 8 h. (A) S. coccinea untreated control, (BD) virescens chlorophyll mutations, (E and F) lethal albina chlorophyll mutations, and (G and H) leaf deformations.

  • Fig. 4.

    Mutants from the M2 population of Salvia coccinea treated with 1.2% ethyl methanesulfonate for 8 h. Pictured on the left is an albina chlorophyll mutation, the center leaf is a chlorina mutation, and the leaf on the right is chlorophyll expression in an untreated Salvia coccinea control.

  • Anderson EG. 1923 Maternal inheritance of chlorophyll in maize Bot Gaz. 76 411 418

  • Baghery MA, Kazemitabar SK & Keari RE. 2016 Effect of EMS on germination and survival of okra (Abelmoschus esculentus L.) Biharean Biol. 10 33 36

  • Bahajantri A & Patil VS. 2013 Studies on ethyl methane sulphonate (EMS) induced mutations for enhancing variability of gladiolus varieties (Gladiolus hybridus Hort) in M1V2 generation Karnataka J Agric Sci. 26 403 407

    • Search Google Scholar
    • Export Citation
  • Branch WD & Brown N. 2019 Inheritance of an albio-virescent leaf mutant in the cultivated peanut (Arachis hypogaea L.) Peanut Sci. 46 203 205 https://doi.org/10.3146/PS19-3.1

    • Search Google Scholar
    • Export Citation
  • Buschmann C. 2007 Variability and application of the chlorophyll fluorescence emission ratio red/far-red of leaves Photosynth Res. 92 261 271 https://doi.org/10.1007/s11120-007-9187-8

    • Search Google Scholar
    • Export Citation
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Rebekah C.I. Maynard Department of Horticulture, University of Georgia, Athens, GA 30602, USA

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John M. Ruter Department of Horticulture, University of Georgia, Athens, GA 30602, USA; and Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA 30602, USA

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Contributor Notes

R.C.I.M. is the corresponding author. E-mail: rebekah.maynard@uga.edu.

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