Introduction

Chinese cabbage (Brassica rapa ssp. pekinensis) is one of the most economically important leafy vegetable crops because it has excellent nutritional qualities and is rich in phytochemicals such as glucosinolates, phenolics, flavonoids, anthocyanins, and carotenoids. The consumption of Chinese cabbage contributes to a reduced risk of diseases such as cancer, diabetes, heart disease, high blood pressure, and osteoporosis (Vanjildorj et al., 2009). Numerous studies have tried to enrich agronomical and nutritional traits of Chinese cabbage using genetic engineering. In vitro plant regeneration and Agrobacterium tumefaciens-mediated transformation methods have been standardized for this crop.

However, the success of these protocols for Chinese cabbage is limited because of its low regeneration efficiency and the poor ability of its explants to regenerate in tissue culture (Mollika et al., 2011). A reliable in vitro regeneration method is necessary to successfully breed economically significant agricultural crops, such as Chinese cabbage. Chinese cabbage cv. Kenshin was used for this study because it is one of the parental lines from the ‘Chiifu-401-42’ and ‘Kenshin-402-43 doubled haploid (CKDH) mapping population and it has a genetic map, which can be considered as a reference for functional genomics (Vanjildorj et al., 2009). Hence, inbred lines, CNU_11635 and rapid-cycling RCBr cv. R-o-18, have been examined using a standardized protocol derived from cv. Kenshin to determine their regeneration potential. The aim of this study was to increase the regeneration potential of Chinese cabbage explants by optimizing the combination of plant growth regulators (PGRs) and other components in the growth media.

Materials and Methods

Plant Material

Mature seeds of B. rapa (cv. Kenshin) were surface-sterilized as described by Vanjildorj et al. (2009). Cotyledon explants were taken from 4-day-old seedlings grown in vitro at 25 ± 2°C under a 16-h-light/8-h-dark photoperiod (irradiance of 50 µmol·m^-2·s^-1). Hypocotyl explants (-1 cm in length) were taken from seedlings grown in vitro in the dark for 7 days and in the light for 3 days. Detached cotyledon explants were positioned with the petiole touching the medium.

Effect of Cytokinins on Multiple Shoot Production

To determine the optimum concentration and type of cytokinin, hypocotyl and cotyledon explants were placed on MS medium (Murashige and Skoog, 1962) without cytokinin (control) or MS medium supplemented with benzyl adenine (BA; 1 - 6 mg·L^-1),1-phenyl-3-(thiadiazol-5-yl)-urea (TDZ; 0.2 - 3.0 mg·L^-1),or zeatin (0.2 - 5 mg·L^-1). The cultures were maintained for 12 weeks and subcultured once at the end of the fourth week. After 12 weeks of culture, the frequency of responding explants, mean number of shoots per responsive explant, mean shoot length, and morphological characteristics were evaluated.

Effect of Cytokinins Combined with Auxins on Multiple Shoot Production

To determine the optimum combination of cytokinins and auxin, both types of explants were transferred to MS medium containing BA (4 and 5 mg·L^-1for hypocotyl and cotyledon explants, respectively) with different concentrations (0.5 - 2.5 mg·L^-1) of indoleacetic acid (IAA), indole-3-butyric acid (IBA), or naphthalene acetic acid (NAA). Based on the results of the preceding experiment, the optimal concentration of BA was selected for testing in combination with the auxins, and MS medium without auxin was used as the control. After 12 weeks of culture, the frequency of responding explants, mean number of shoots per responsive explant, mean shoot length, and morphological characteristics were evaluated.

Effect of AgNO₃ on Multiple Shoot Production

The two types of explants were transferred to MS medium containing BA and NAA (4 mg·L^-1+1 mg·L^-1 and 5 mg·L^-1+0.5 mg·L^-1 for hypocotyl and cotyledon explants, respectively) and AgNO₃ at different concentrations (1 - 5 mg·L^-1) to increase the production of multiple shoots. Here, based on the preceding experiments, the optimal concentrations of BA and NAA were combined with AgNO₃, and MS medium without AgNO₃ was used as the control. After 12 weeks of culture, the frequency of responding explants, mean number of shoots per responsive explant, mean shoot length, and morphological characteristics were evaluated.

Effect of Type and Concentrations of Auxins on Root Induction

After 12 weeks of culture, shoots longer than 3 cm that regenerated from hypocotyl and cotyledon explants were transferred to MS medium containing IBA, IAA, or NAA (0.5 - 2.0 mg·L^-1) and cultured for 4 weeks for root induction. In this experiment, MS medium without auxin was used as the control. After 4 weeks of culture, the frequency of responding shoots, mean number of roots per responsive shoot, and mean root length were recorded.

Effect of Media Strength on Root Induction

To determine the optimal strength of medium for root induction, in vitro-regenerated shoots (-3 cm in length) from both types of explants were transferred onto MS medium at several different strengths (1, 0.75, 0.50, and 0.25) without auxins. After 4 weeks of culture, the frequency of responding shoots, mean number of roots per responsive shoot, and mean root length were recorded. All cultures were incubated at 25 ± 2°C under a 16-h-light/8-h-dark photoperiod (irradiance of 50 µmol·m^-2·s^-1).

Acclimatization

After 4 weeks, the rooted plants were washed carefully in running tap water to remove the agar and then transferred to a plastic container containing autoclaved perlite. The plants were grown in a plant growth room at 75% relative humidity for 7 weeks and then moved to a glasshouse for the hardening process. Initially, all the plants were covered with polyethylene bags to maintain high humidity and supplied with water daily. The polyethylene bags were slowly removed when the plants showed signs of acclimatization. The plants were subsequently transferred to a glasshouse.

Statistical Analyses

After optimizing the regeneration methods for cv. Kenshin using different concentrations and combinations of PGRs along with media strength, inbred lines, CNU_11635 and RCBr cv. R-o-18, were tested for regeneration ability using the best hormonal concentrations and media strength. A completely randomized design was used for this study. The tests were repeated three times, each with three replicates. Data shown are mean ± standard error (SE). Mean separations were carried out using Duncan’s multiple range test, and significance was determined at the 5% level (SPSS 17.5)

Results and Discussion

Effect of Cytokinins on Multiple Shoot Production

Hypocotyl and cotyledon explants cultured on control medium (MS basal medium) did not form meristem or axillary buds and eventually died (Suppl. Tables 1s and 3s).The inclusion of PGRs in the media favored meristematic cell development and shoot bud initiation, resulting in shoot regeneration (Suppl. Tables 1s and 3s). The cytokinin type and its concentration affected the average number of shoots per cultured explant type and length of shoot per explant. The morphogenic responses of hypocotyl and cotyledon explants are depicted in Suppl. Tables 1s and 3s. The cotyledon explants cultured on MS medium containing cytokinin started extending and swelling after 2 weeks of culture (Fig. 1A), whereas hypocotyl explants became distended after 7 days of culture (Fig. 2A). The two types of explants did not show the same regeneration abilities.

Fig. 1.

In vitro regeneration of shoots from cotyledon explants of Brassica rapa. A. Initiation of meristematic cells at the petiole detached region in cotyledon explant at 2 weeks of culture (scale bar = 0.25 cm). B. Induction of shoot primordia from differentiated meristematic cells at the petiole detached region in cotyledon explant at 4 weeks of culture (scale bar = 0.25 cm). C and D. Dedifferentiation of shoots from cotyledon explant after 6 weeks of culture (scale bar = 0.25 cm). E, F, and G. Proliferation of shoots from cotyledon explant after 5, 6, and 7 weeks of culture, respectively (scale bar = 0.5 cm). H, I, and J. High proliferation of multiple shoots from cotyledon explants after 8, 10, and 12 weeks of culture, respectively (scale bar = 0.5 [H and I] and 1cm [J]). K. Production of multiple shoots from cotyledon explants after 16 weeks of culture (scale bar = 1 cm). L.Growth of roots regenerated from elongated shoots after 4 weeks of culture. All the shoot cultures were grown on MS medium supplemented with 5 mg·L^-1 BA, 0.5 mg·L^-1 NAA, and 2 mg·L^-1 AgNO₃, and root induction was obtained on half-strength MS basal medium (scale bar = 1 cm).

Fig. 2.

In vitro regeneration of shoots from hypocotyl explants of Brassica rapa. A. Bulging of explants after 1 week of culture (scale bar = 0.25 cm). B. Initiation of meristematic cells with hairy-like structure after 3 weeks of culture (scale bar = 0.25 cm). C. Induction of shoot differentiation at the cut end of a hypocotyl explant after 5 weeks of culture (scale bar = 0.25 cm). D, E, and F. Proliferation of shoots from a hypocotyl explant after 6 and 12 weeks of culture (scale bar = 0.25 cm). G and H. Well-proliferated multiple shoots from hypocotyl explant on the 16th week of culture (scale bar = 1 cm). I. Profusely developed roots in an elongated shoot after 4 weeks of culture (scale bar = 1 cm). All the shoot cultures were grown on MS medium supplemented with 4 mg·L^-1 BA, 1 mg·L^-1 NAA, and mg·L^-1 AgNO₃, and root induction was obtained on half-strength MS basal medium.

After 28 days of culture, a mass of regenerative meristematic cells originated from the cut end of the petiole of the cotyledon explants (Fig. 1B). These cells were whitish in color at the initial stage of differentiation and became greenish-white during subsequent growth. These stages were observed in cotyledon explants on media containing BA at all concentrations except for0.5 and 1 mg·L^-1. After 21 days of culture, a mass of parenchyma cells formed at the cut end of hypocotyl explants and turned green (Fig. 2B). These cells were responsible for shoot bud initiation. Among the various cytokinins tested, BA at 5 or 4 mg·L^-1in MS medium was the most effective for promoting multiple shoot production (mean, 1.64 shoots/cotyledon explant and 1.47 shoots/hypocotyl explant, respectively, after 12 weeks (Suppl. Tables 1s and 3s). Although multiple shoot buds formed on media containing BA and TDZ at all analyzed concentrations, multiple shoot formation and the highest rates of shoot regeneration from both explant types were recorded on MS medium containing BA (Suppl. Tables 1s and 3s).

During the tissue culture process, BA is degraded and conjugates with inert compounds (Kamínek, 1992). In previous studies, BA was found to strongly affect the shoot regeneration efficiency of Brassica napus (Tang et al., 2011), Brassica juncea (Guo et al., 2005), and Brassica oleracea (Gerszberget al., 2015). Burnett et al. (1994) obtained 32 shoots/B. rapa ssp. oleifera cotyledon explant on MS medium supplemented with BA (1 mg·L^-1). Radke et al. (1992) recorded a 43% shoot response (at least one shoot produced per explant) of B. rapa ssp. oleifera cotyledon explants on MS medium supplemented with BA (3 mg·L^-1), zeatin (1 mg·L^-1), andAgNO₃ (5 mg·L^-1). Cogbill et al. (2010) obtained 20 shoots/B. rapa (RCBr) cotyledon explant on MS medium supplemented with TDZ (1.5 mg·L^-1), NAA (0.5 mg·L^-1), and AgNO₃ (5 mg·L^-1). Teo et al. (1997) also obtained 20 shoots/B. rapa (RCBr) cotyledon explant on MS medium supplemented with BA (4.5 mg·L^-1) and NAA (0.4 mg·L^-1). Yang et al. (2004) obtained high shoot regeneration efficiency (40%) by culturing cotyledon explants of B. rapa ssp. pekinensis on medium containing BA (5 mg·L^-1) and NAA (0.5 mg·L^-1). In this study, we obtained higher numbers of multiple shoots from both types of explants in terms of mean number of shoots and percentage of responses (Suppl. Tables 1s and 3s).

In this study, the number of multiple shoots and shoots regeneration responses of cotyledon and hypocotyl explants increased by optimizing the concentration of cytokinin. Multiple shoot regeneration decreased as BA concentration increased beyond the optimal concentration (Suppl. Tables 1s and 3s). Carmen et al. (2001) postulated that exogenously applied cytokinins play a major role in promoting axillary meristem formation, as they support the propagation of meristematic cells in the axillary buds and boost the number of bud primordia from the pre-existing meristems. Here, we obtained a large number of multiple shoots in a short time via axillary bud formation from cotyledon and hypocotyl explants. The analysis of variance showed that the mean shoot number and the response rate were significantly affected by the type and concentration of cytokinin. BA induced in vitro development of meristems most effectively from both explant types of B. rapa.

Effect of Cytokinin and Auxins on Multiple Shoot Production

To further optimize shoot production, cotyledon and hypocotyl explants were cultured on media containing three types of auxins (IAA, IBA, and NAA) at different concentrations (0.5 - 2.5 mg·L^-1) with optimal concentrations of BA (5 and 4 mg·L^-1for cotyledon and hypocotyl explants, respectively). Of the three different auxins tested in combination with BA, NAA was the most effective for the induction and subsequent proliferation of multiple shoots from both types of explants. Including NAA with BA in the medium significantly increased the shoot number, compared to that obtained using other auxins, suggesting that there was a synergistic/additive effect of NAA with BA on shoot induction and proliferation. Auxins have been demonstrated to have synergistic, antagonistic, and additive interactions with cytokinins at multiple levels (depending on the plant species and tissue type) in the regulation of physiological responses (Coenen and Lomax, 1997).

The active cytokinin concentration in plants can be regulated by auxin and vice versa. In addition, auxin regulates cytokinin biosynthesis more strongly than cytokinin regulates auxin biosynthesis (Nordström et al., 2004). Among the different auxins, NAA is the only one that does not require active uptake to pass through the plasma membrane into plant cells (Nordström et al., 2004). This may explain why we observed the maximum frequency (68%) of multiple shoot induction on MS medium supplemented with NAA (0.5 mg·L^-1) and BA (5 mg·L^-1). In that treatment, the mean number of shoots/cotyledon explant was 1.77 and the average shoot length was 7.24 cm after 12 weeks of culture (Suppl. Tables 2s and 4s). Similarly, the highest overall number of shoots (2.86 shoots/hypocotyl explant) and maximum response (76%) were recorded for hypocotyl explants on MS medium containing NAA (1 mg·L^-1) supplemented with BA (4 mg·L^-1) after 12 weeks of culture (Suppl. Tables 2s and 4s). Liu et al. (2018) obtained 1.8 shoot/leaf explant of B. rapa cv. Beijing New No. 3 on MS medium supplemented with BA (4 mg·L^-1) and NAA (1 mg·L^-1). In this study, 1.5-fold improvement was recorded in shoot regeneration of B. rapa cv. Kenshin. Exogenous auxins and cytokinins may affect endogenous hormone levels in the explants (Sivanandhan et al., 2011). The shoot regeneration efficiency was increased by a combination of BA and NAA in B. rapa. Compared to NAA, the other auxins tested in these experiments did not favor multiple shoot production. Increasing or decreasing auxin levels in the culture medium above or below optimum levels negatively affected multiple shoot proliferation. Higher concentrations of auxin that exceeded the optimal level adversely affected the regeneration potential of both explants. Excess exogenous or endogenous auxins may negatively affect regeneration potential. In another study, excess auxin affected meristematic cell division, resulting in the decreased growth responses (Wernicke and Milkovitz, 1987).

Effect of AgNO₃ on Multiple Shoot Production

In this study, AgNO₃promoted shoot regeneration from both types of B. rapa. Shoots formed more rapidly, and were larger, when explants were cultured on MS medium containing AgNO₃ and PGRs. The optimum concentration of AgNO₃ for regeneration differed depending on the explant type. A lower concentration of AgNO₃ (2 mg·L^-1) along with optimum concentrations of BA and NAA resulted in significantly more shoots per cotyledon explant (2.38/explant; Table 2 and Fig. 1) with a 74% regeneration response. A higher concentration of AgNO₃ (4 mg·L^-1) was required for maximum shoot induction from hypocotyl explant (3.41 shoots/explants; Table 1) with a 79% regeneration response (Fig. 2). Concentrations of AgNO₃ above or below the optimal concentration in the medium resulted in minimal regeneration of both explants types (Tables 1 and 2). Liu et al. (2018) recorded 2.02 shoots/leaf explant of B. rapa cv. Beijing New No. 3 on MS medium supplemented with BA (4 mg·L^-1), NAA (1 mg·L^-1),and AgNO₃ (2 mg·L^-1). In this study, 1.68-fold enhancement was noted in shoot regeneration of B. rapa cv. Kenshin. Zhang et al. (1998) obtained higher number of shoots from cotyledon explants of B. campestris upon addition of AgNO₃ at 2 mg·L^-1.

Table 1. Effect of AgNO₃ in combination with BA and NAA on in vitro regeneration of shoots from hypocotyl explants derived from 10-day-old in vitro seedlings of Brassica rapa after 12 weeks of culture. The whole experiment was performed with 4 mg·L^-1 BA and 1 mg·L^-1 NAA with AgNO₃

Table 2. Effect of AgNO₃ in combination with BA and NAA on in vitro regeneration of shoots from cotyledon explants derived from 4-day-old in vitro seedlings of Brassica rapa after 12 weeks of culture. The whole experiment was performed with 5 mg·L^-1 BA and NAA 0.5 mg·L^-1 with AgNO₃

Cogbill et al. (2010) reported that 5 mg·L^-1AgNO₃increased the number of shoots in cultures of rapid-cycling B. rapa along with TDZ and NAA. In this study, AgNO₃did not increase the shoot formation response to TDZ in either explant type (data not shown). Teo et al. (1997) observed a drastic inhibition of shoot regeneration of rapid-cycling B. rapa on MS medium supplemented with AgNO₃. These differences among studies emphasize the genotypic specificity of responses during in vitro regeneration. Our results showed that including AgNO₃inMS medium in combination with BA and NAA promoted shoot regeneration in B. rapa.

Effect of Type and Concentration of Auxins on Root Induction

As noted by Sivanandhan et al. (2011), efficient acclimatization is important for establishing in vitro-derived plants in the field, and this process requires the development of roots from elongated shoots. In our study, root initiation was visible after 15 days of culture in elongated shoots regenerated from hypocotyl explants. However, shoots that regenerated from cotyledon explants took 20 days to form roots on MS medium containing auxins. There was a noticeable difference in rooting efficiency between the two types of B. rapa explants. This may be because of differences in endogenous hormone concentrations or the balance of hormones, the age of explants, or differences in their sensitivity to external PGRs. The entire tissue culture process, including the control (without PGRs) favored root induction. However, the percentage of rooting responses and physiology of the roots varied depending on the types and concentrations of PGRs. The highest rooting percentage (88%) and maximum number of roots (9 roots/shoot and average root length of 10 cm/shoot) were obtained from hypocotyl-derived shoots after 4 weeks of culture on MS medium containing NAA (2 mg·L^-1) (Suppl. Table 5s).

Shoots from cotyledon explants grown on medium containing 0.5 mg·L^-1NAA had an average root length of 9 cm, and 85% of these shoots formed roots (Suppl. Table 6s). For cotyledon explants, the best rooting response (8 roots per shoot) was on MS medium containing 0.5 mg·L^-1NAA. There were no significant differences between the two types of explants (Suppl. Tables 5s and 6s). Compared with NAA, the other auxins did not promote rooting from regenerated and elongated shoots as effectively (Suppl. Tables 5s and 6s). The juvenile roots were flimsy and pale but had darkened in color and become stranger by the end of the fourth week of culture. Burnett et al. (1994) analyzed root formation in B. rapa ssp. oleifera cultured on MS basal medium but did not describe the rooting process in detail. Teo et al. (1997) found that rapid-cycling B. rapa showed greater rooting responses on MS medium containing NAA than on MS basal medium without PGRs and obtained three roots/shoot and a 49% rooting response on medium containing 1 µM. In this study, the strongest rooting response was on medium containing NAA at 2 mg·L^-1. Cogbill et al. (2010) obtained a rooting response of 28% for B. rapa (RCBr) on MS medium containing 0.5 mg·L^-1NAA. Abbasi et al. (2011) found that B. rapa var. turnip showed the strongest rooting response (72%) and formed six roots/shoot on MS medium containing IBA at 3 mg·L^-1. Liu et al. (2018) reported that B. rapa showed a good rooting response on MS medium containing 0.6 mg·L^-1NAA. These differences among the studies might be due to different genotypes of plants, different explant types, and/or different environmental conditions.

Effect of Media Strength on Root Induction

To increase the number of roots regenerating from elongated shoots from both hypocotyl and cotyledon explants, we tested MS media at different strengths. Among the different strengths tested, half-strength MS medium resulted in the best rooting response (92%) with the maximum root number (14 roots/elongated shoot) and greatest root length (11 cm/shoot) of roots from shoots derived from hypocotyl explants (Table 3, Fig. 2I and Suppl. Fig. 2s A). On the same medium, elongated shoots derived from cotyledon explants had a similar root formation response (90%) with slightly fewer roots per shoot (11 roots/elongated shoot) and slightly shorter root length (10 cm/shoot) after 4 weeks of culture (Table 4, Fig. 2L and Suppl. Fig. 1s A). The rooting response was not significantly different between hypocotyl and cotyledon explants but was better on half-strength MS medium than on full-strength MS medium or medium supplemented with auxin (Suppl. Tables 5s and 6s). Sivanandhan et al. (2011) reported that the number and length of roots on in vitro-regenerated shoots were key factors for successful hardening and acclimatization. By optimizing the rooting medium, we achieved 100% acclimatization and survival of in vitro plants after transplanting into the field.

Table 3. Effect of media strength on root formation from in vitro-raised elongated shoots regenerated from hypocotyl explants of Brassica rapa after 4 weeks of culture

Table 4. Effect of media strength on root formation from in vitro-raised elongated shoots regenerated from cotyledon explants of Brassica rapa after 4 weeks of culture

Conclusions

Our results showed that hypocotyl explants of B. rapa are a good starting material for in vitro propagation. The combination of BA (4 mg·L-1), NAA (1 mg·L-1), and AgNO3 (4 mg·L-1) was optimal for the regeneration of multiples hoots from explants in in vitro culture. Exogenous BA and NAA interacted with AgNO3 in a synergistic/additive manner to promote multiple shoot production and proliferation and thus enhanced the number of multiple shoots by 1.68-fold. Root formation was optimal on half-strength MS medium without PGRs. After acclimatization, the plants grew normally in greenhouse conditions. The results of this study will be useful for regenerating plants after gene transfer and for genome editing.

Supplementary Material

Supplementary materials are available at Horticultural Science and Technology website (https://www.hst-j.org).