Leaf-induced callus formation in two cultivars: hot pepper ‘CM334’ and bell pepper ‘Dempsey’

ABSTRACT

Pepper (Capsicum annuum), one of the most economically important vegetables of the Solanaceae family, is cultivated worldwide. To apply versatile genome-editing tools to a pepper genome for precise molecular breeding, an in vitro regeneration protocol is indispensable and callus formation is an essential step in the regeneration of pepper. Here, we show that calli were successfully induced from young leaves (3–4 cm) of pepper plants, the hot pepper C. annum ‘CM334’ (‘CM334’) and bell pepper C. annum ‘Dempsey’ (‘Dempsey’), grown on soil for less than 7 weeks. The excised leaf segments of ‘CM334’ produced white calli in B5 medium containing 3% sucrose (3S), 2 mg/L 6-benzylaminopurine (2BAP), and 1 mg/L α-naphthalene acetic acid (1NAA). The calli were able to proliferate in B5 3S 2BAP medium supplemented with 2-morpholinoethanesulphonic acid (MES) and 1.5 mg/L NAA (1.5NAA). The excised leaf segments of ‘Dempsey’ produced light-yellow and friable calli in MS medium supplemented with B5 vitamins (MSB5), 3S and 1 mg/L 2,4-dichlorophenoxyacetic acid (1 2,4D), and the calli were also maintained in the same medium. Our findings establish the conditions for leaf-derived callus formation, which is the basis for regeneration of whole plants for two different pepper cultivars, for obtaining stable protoplasts, and eventually for applying genome-editing tools to improve the quality of peppers.

KEYWORDS:

• C. annum ‘CM334’
• C. annum ‘Dempsey’
• Callus formation
• pepper protoplasts

Pepper of the Solanaceae family presents difficulties in terms of in vitro tissue culture and organ differentiation.¹ Capsicum annuum is the most widespread and cultivated species in sub-tropical and temperate countries. The whole genomic sequence of C. annuum ‘CM334’ was released in 2014,² and the genetic study and molecular breeding program for improving pepper have become more efficient and easier similar to that for other Solanaceae species such as tobacco³ and tomato.⁴ Although the pepper genome sequence is available,^2,5 the biggest hurdle for the functional study and improving the quality of pepper is in vitro tissue culture and regeneration. The difficulty in obtaining enough seeds of the pungent cultivar ‘CM334’ limits the application of biotechnological tools for improving its quality. Moreover, the leaf-derived protoplasts of ‘CM334’ were very sticky and prone to collapse, making it difficult to conduct a cell biological study for validating the best tool kit for genome editing. Thus, it is extremely vital to have enough materials for cell biological and functional genetic studies pertaining to the improvement of pepper quality. In pepper, leaf-derived callus formation can be a central platform not only to develop an optimal condition for regeneration but also to provide a stable pepper protoplast system for applying genome editing tools.⁶

Few cases have been reported for successful regeneration of peppers.^7,8 However, there are still many constraints in the successful regeneration of peppers. The regeneration showed genotype-dependency and variation in the effect of phytohormones.⁹ In general, for in vitro tissue culture, demands for seeds are high due to the use of young tissues such as cotyledons, seedlings, hypocotyls, and embryonal explants.¹⁰ Here, we used a soil-grown and surface-sterilised leaf as an explant, instead of using tissues derived from young plants, thus eliminating the need of a large number of seeds. We provided optimal conditions for the formation of leaf segment-derived calli from the genome-sequenced hot pepper (‘CM334’) and bell pepper (‘Dempsey’). To figure out the callus-inducing conditions, we used leaf tissues (3–4 cm) from plants grown on soil less than 7 weeks, sterilised the leaf surface with 70% ethanol for 5 min, and subsequently rinsed it three times with sterile distilled water. The basic media for leaf-induced callus formation were B5 or MSB5 medium containing 3% sucrose (3S).

To define the conditions for callus induction, sterilised ‘CM334’ leaf discs (0.5–1 cm) were placed with the abaxial surface upward on B5 media containing 3S and 2 mg/L BAP together with 0.5, 1.0, or 1.5 mg/L NAA. All of the tested conditions, B5, 3S, 2 mg/L BAP medium containing 0.5 NAA, 1 NAA, or 1.5 mg/L NAA, were able to induce calli from leaf discs successfully. However, of these tested conditions, the optimal condition was B5 medium supplemented with 3S, 2 mg/L BAP and 1 mg/L NAA (Figure 1a, 1b) (Figure S1a, Table1). The leaf-derived micro-calli were detached from the leaf discs and transferred on the same medium for calli proliferation. Two consecutive subcultures of the micro-calli in B5 3S 2BAP 1NAA caused the calli to turn brown and reduced the proliferation activity. When MES medium containing B5 3S 2BAP 1.5NAA was used the leaf-derived calli propagated properly without the colour change to brown (Figure 1c). The calli with the ability to proliferate properly were successfully maintained more than 8 months after initial callus induction. The maintained calli provided sustainable protoplasts without prone to collapse (Figure 1d).

Table 1. Summary of the callus properties of the two pepper cultivars ‘CM334’ and ‘Dempsey’ depending on the callus induction media.

Cultivar	Media	Callus formation efficiency	Callus growth rate	Callus colour
CM334	B5 2BAP 0.5 NAA	100%	+	Light to dark yellow, friable
	B5 2BAP 1.0 NAA	100%	++	Light-yellow, friable
	B5 2BAP 1.5 NAA	100%	+++	Light to dark yellow, friable
Dempsey	B5 2BAP 0.5 NAA	70%	++	White and hard
	B5 2BAP 1.0 NAA	90%	++	White and hard
	B5 2BAP 1.5 NAA	35%	+	White and hard
	MSB5 1 2,4D	80%	+++	Light-yellow and friable

Callus formation efficiency (%) was calculated by dividing the number of callus-formed leaf segments by the number of total leaf segments (n > 200).

Figure 1. Leaf-induced callus formation of Capsicum annum ‘CM334’ and ‘Dempsey’. (a-c) Leaf segment-derived calli of ‘CM334’. (a) 4-week-old leaf segments placed in Gamborg’s B5 medium supplemented with 2 mg/L 6-benzylaminopurine (2BAP), and 1 mg/L α-naphthalene acetic acid (1NAA) medium under long-day conditions (16 h light/8 h dark) at 25°C in a growth chamber. (b) Enlarged image of (a). (c) Propagated calli in B5 medium containing 2BAP, 1.5 mg/L NAA, and 2-morpholinoethanesulphonic acid (MES) under dark conditions at 25°C. (d) Protoplasts isolated from the propagated ‘CM334’ calli. (e-g) Leaf segment-derived calli of ‘Dempsey’. (e) 4.5-week-old leaf segments placed in Murashige and Skoog (MS) medium supplemented with B5 vitamins and 1 mg/L 2,4-dichlorophenoxyacetic acid (1 2,4D) under long-day conditions (16 h light/8 h dark) at 25°C in a growth chamber. (f) Enlarged image of (e). (g) Calli propagated in MSB5 (MS basal salt mixture and B5 vitamins) medium containing 1 2,4D under dark conditions at 25°C. (h) Protoplasts isolated from the propagated ‘Dempsey’ calli. White scale bars = 1 cm. Black scale bars = 10 μm.

In the case of the bell pepper ‘Dempsey’, the surface-sterilised leaves were incubated in the same media as ‘CM334’; B5 3S 2BAP with NAA ranging from 0.5 to 1.5 mg/L NAA. The ‘Dempsey’ leaf segments were able to produce micro-calli of various sizes, which were inversely correlated with the concentration of NAA included in the medium. The micro-calli were propagated as white and hard calli, which were not suitable for further subculture (Figure S1B, Table1). When the excised leaves of ‘Dempsey’ were incubated with MSB5 medium supplemented with MES and 1 mg/L 2,4 D, leaf-derived micro-calli were light-yellow and friable (Figure 1e, 1f). Subsequently, the friable micro-calli of ‘Dempsey’ were able to proliferate and maintained more than 8 months in the same medium, MSB5 3S 1 2,4D, without a colour change to brown (Figure 1g). The ‘Dempsey’ calli also provided copious protoplasts like ‘CM334’ calli (Figure 1h). Taken together, leaf-derived callus formation from peppers, ‘CM334’ and ‘Dempsey’ whose genomic sequences have been known, would be a starting point for precise genome editing to improve the quality of pepper via cell-based molecular studies and in vitro tissue culture, and regeneration.

Abbreviations

Table

B5	Gamborg’s B5 medium
BAP	6-benzylaminopurine
NAA	α-naphthalene acetic acid
MES	2-morpholinoethanesulphonic acid
MS	Murashige and Skoog medium
2,4 D	2,4-dichlorophenoxyacetic acid
MSB5	medium consisting of the MS basal salt mixture and B5 vitamins
S	Sucrose

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Byoungcheorl Kang for sharing peppers; Two cultivars, ‘CM334’ and ‘Dempsey’, were provided by the Vegetable Breeding Research Center. We thank Jisun Choi for technical assistance; Hyunah Lee of the Central Laboratory of Kangwon National University for LSM880 microscopy. We also thank all other members of the PCGE lab.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by 2018 Research Grant from Kangwon National University; the Basic Science Research Program (grants NRF-2017R1C1B5076421 and NRF-2018R1A2B6006233); and the X-mind Corps program (2017H1D8A1028271) of the National Research Foundation of Korea (NRF) funded by the Korean Government (MSIT).