A Lower pH Value Benefits Regeneration of Trichosanthes kirilowii by Somatic Embryogenesis, Involving Rhizoid Tubers (RTBs), a Novel Structure

A new approach was established for the regeneration of Trichosanthes kirilowii from root, stem, and leaf explants by somatic embryogenesis (SE), involving a previously unreported SE structure, rhizoid tubers (RTBs). During SE, special rhizoids were first induced from root, stem, and leaf explants with average rhizoid numbers of 62.33, 40.17, and 11.53 per explant, respectively, on Murashige and Skoog (MS) medium (pH 4.0) supplemented with 1.0 mg/L 1-naphthaleneacetic acid (NAA) under dark conditions. Further, one RTB was formed from each of the rhizoids on MS medium (pH 4.0) supplemented with 20 mg/L thidiazuron (TDZ) under light conditions. In the suitable range (pH 4.0–9.0), a lower pH value increased the induction of rhizoids and RTBs. Approximately 37.77, 33.47, and 31.07% of in vivo RTBs from root, stem, and leaf explants, respectively, spontaneously developed into multiple plantlets on the same MS medium (supplemented with 20 mg/L TDZ) for induction of RTBs, whereas >95.00% of in vitro RTBs from each kind of explant developed into multiple plantlets on MS medium supplemented with 5.0 mg/L 6-benzylaminopurine (BAP). Morphological and histological analyses revealed that RTB is a novel type of SE structure that develops from the cortex cells of rhizoids.

regeneration through SE has not been established. In this study, with the optimization of pH values and concentrations of plant growth regulators (PGRs), a high-efficiency regeneration system was established in T. kirilowii, and rhizoid tubers (RTBs), a novel SE structure, were first observed and named. To our knowledge, this type of SE structure has not previously been reported. It was also found that lower pH values of a medium could significantly promote the induction of rhizoids and RTBs, and further contribute to the high efficiency of SE and regeneration in T. kirilowii.

Results
Rhizoids were first induced from stem, leaf, and root explants in T. kirilowii by adding NAA to the medium; lower pH values significantly promoted rhizoid induction. Two auxin analogues, NAA and 2,4-D, with a concentration series of 0, 0.5, 1.0, and 1.5 mg/L, were used to optimize PGR conditions for the induction of rhizoids. Without NAA and 2,4-D in the medium, no rhizoids were induced ( Table 1), suggesting that adding PGR is necessary for rhizoid induction. For all of the 2,4-D supplementary concentrations, no rhizoids were induced, indicating that 2,4-D is not suitable for rhizoid induction in T. kirilowii. For the NAA supplements, a concentration of 1.0 mg/L resulted in significantly higher average numbers of induced rhizoids per explant from the root, stem, and leaf explants (31.03, 18.90, and 8.03, respectively) ( Table 1) than other tested concentrations. For the supplementary NAA concentrations of 0.5, 1.0, and 1.5 mg/L, the rates of rhizoid induction from different explants exhibited the following sequence: root . stem . leaf. With the optimal NAA supplement, three weeks after the inoculation of three types of explants (root, stem, and leaf) ( Fig. 1 A, B, and C), small white rhizoids were induced from all three types of explants and developed into a cluster of rhizoids surrounded by many hair-like bodies ( Fig. 1 A1, B1, and C1).  Table 2). Media of pH 3.0 could not be solidified, and no rhizoids were induced. Root, stem, and leaf explants on media of pH 4.0 had the highest numbers of induced rhizoids per explant (62.33, 40.17, and 11.53, respectively) ( Table 2). Except for pH 3.0, which gave no rhizoid induction, other pH values exhibited the following sequence for the average numbers of induced rhizoids per explants: 4.0 . 5.0 . 6.0 . 7.0 . 8.0 . 9.0 ( Table 2) for each type of explant. This suggests that, within a suitable range of pH values (pH 4.0-9.0), a lower pH value increases the induction of rhizoids in T. kirilowii.
RTBs were induced from rhizoids by adding TDZ to media and lower pH values significantly promoted induction. Rhizoids induced from MS media (pH 5.8) with an optimal supplement of NAA (1.0 mg/L) were transferred to MS media (pH 5.8) containing TDZ to form RTBs. Compared with 10 and 30 mg/L TDZ supplements, the 20 mg/L TDZ supplement resulted in significantly higher RTB induction rates for root, stem, and leaf explants (Table 3), for which   Identification and morphological analysis of RTBs. Double staining with acetocarmine and Evans blue was employed to analyse whether the induced RTBs were mainly composed of embryogenic tissue. With double staining, embryogenic tissue, in which cells reproduce and metabolize quickly, was stained by both dyes and appeared bright red, while non-embryogenic tissue, in which cells could not be stained with acetocarmine because of their lower metabolism and reproduction, was stained only by Evans blue and appeared blue. This showed that the induced RTBs were in the shape of a ball and were mainly composed of embryogenic tissue in red with a small amount of non-embryogenic tissue in dark blue on the surface (Fig. 2 A1, A4, and A5; B1, B4, and B5; C1, C4, and C5), suggesting that RTB is a kind of special SE structure with the potential to develop into stem embryos. To our knowledge, this is the first observation of RTB; therefore, it is considered as a novel SE structure. By using microscopic squash technology, together with DAPI staining (    embryogenic cells of RTBs were observed to be closely arranged with a thick cytoplasm occupying a large part of the RTB, while nonembryogenic callus cells were much looser in arrangement than embryogenic cells (Fig. 2 A2, B2, and C2). This further confirmed that RTBs are a kind of SE structure.
Internal structures of RTBs. In order to analyse the internal structure of RTBs, free-hand slices demonstrating transverse and longitudinal sections of RTBs were directly observed and stained with borax-toluidine blue for further observation of the arrangement of RTB cells. The direct observation of slices without staining showed that embryoids were initially induced from the cortex cells of RTBs ( Fig. 3 A, A1, B, and B1), it also demonstrated that multiple embryoids were formed and closely arranged in RTBs at different developmental stages, and developed with the development of RTB ( Fig. 3 C, C1, D, D1, E, E1, F, and F1). At the late stage of development, multiple papillae were formed on the surface of RTBs (Fig. 3 G), and freehand slices of the late-stage RTBs revealed that the papillae were composed of developed and germinated embryoids (Fig. 3 H). The slices were stained with borax-toluidine blue, which caused non- . RTB proembryos started to appear 12 days after rhizoids were transferred to RTB induction medium (Fig. 6 C and E). RTB embryoids started to be formed 14 days after rhizoids were transferred to RTB induction medium (Fig. 6 D and F) and the formed globular, heart, and heart-torpedo embryos grew along the marginal zone of the cortex (Fig. 6 D, F, G, and H). This indicated that RTB embryoids were derived from the cortex cells of rhizoids and sequentially induced, exhibiting different sizes (Fig. 6 A, B, C, and D). It also demonstrated that one individual RTB could contain multiple embryoids at different developmental stages (Fig. 6 G and H).
Plantlets could develop from in vitro and in vivo RTBs. Two methods were used to induce plantlets from in vitro and in vivo RTBs in T. kirilowii. In vivo RTBs on the same induction media as that for inducing RTBs could spontaneously develop into multiple plantlets at a lower frequency, approximately 37.77%, 33.47%, and 31.07% for RTBs from root, stem, and leaf explants, respectively. In order to increase the frequency of plantlet induction, in vitro RTBs  separated from explants were transferred to MS media supplemented with BAP ( Table 5). The result of BAP optimization indicated that in vitro RTBs on MS medium supplemented with 5.0 mg/L BAP developed into multiple plantlets ( Fig. 7) with significantly higher frequencies for root, stem, and leaf explants (97.57%, 95.90%, and 95.97%, respectively) than those on media with other concentrations of BAP (Table 5). The result that one individual RTB could develop into multiple plantlets is different from the usual scenario of SE, in which one SE structure often individually develops and forms one plantlet 26,27 .

Discussion
The optimal supplement of PGRs is important for the success of SE and regeneration in plants 23,28,29 . In the present study, SE in T. kirilowii was achieved through two steps supplemented with NAA and TDZ, respectively. For the first step, rhizoids were induced by adding NAA at an optimal concentration in media, and for the following step RTBs were induced from the rhizoids by adding TDZ at an optimal concentration in media. Auxin analogues of NAA and 2,4-D were tested for rhizoid induction, and 2,4-D did not induce rhizoids in T. kirilowii, whereas NAA resulted in successful rhizoid induction. This suggests that different auxin analogues have different effects on rhizoids in T. kirilowii. Therefore, the right selection and concentration optimization of PGRs is a prerequisite for successful SE. Light conditions also played an important role in the two steps of SE in T. kirilowii; the explants were kept in the dark for rhizoid induction, whereas high light benefited the induction of RTBs. High light conditions are different from those of previous reports in which moderate light was employed for inducing somatic embryos 27,30 . In this paper, we found that a lower pH value (4.0) surprisingly resulted in significantly higher rates for the inductions of rhizoids and RTBs than the normally adopted pH value (5.8), and, for the pH value series of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, increasing pH decreased the induction rates of rhizoids and RTBs. However, the inductions of rhizoids and RTBs failed on media with pH 3.0; it seems that this is too low to induce rhizoids and RTBs. We also found that media with pH 3.0 could not be solidified, which could be one reason for the failure to induce rhizoids and RTBs on media with pH 3.0. This suggests that a lower pH value benefits rhizoid and RTB induction in T. kirilowii within a suitable pH range. The mechanism causing these benefits in T. kirilowii and whether it is universal in other plants need further study. For rhizoids and RTBs originated from root, stem, and leaf explants of T. kirilowii, induction frequencies were significantly different in the sequence: root . stem . leaf. This suggests that different organs have different capacities for rhizoid and RTB induction, and root explants are a good choice for SE via rhizoid and RTB induction in T. kirilowii. The capacity ranking among organs of SE via RTB in T. kirilowii is different from that via frog egg-like bodies in Solanum nigrum (leaf . root . stem) 23 , suggesting that the organ resulting in the highest induction frequency of SE could be different in different SE induction pathways and different plant species. The observed RTBs are considered to be a novel kind of SE structure for the following reasons: 1) to our knowledge, a tuber-shaped structure (RTB) during SE has not previously been reported; 2) evidence showed that RTBs are composed of embryogenic cells that could developed into embryos; 3) rhizoids first formed in SE mediated by RTB, different from the callus formation in a typical SE; 4) multiple embryos can be formed within an individual RTB, whereas embryos are often individually formed on the callus surface in a typical SE pathway; and 5) RTBs can spontaneously develop into multiple plantlets at a higher frequency than embryos in a typical SE pathway. In addition, we found that mature RTBs had a hard, green outside layer. The layer benefits embryo formation and protects embryos inside, which may play a similar role to the protection layer of artificial seeds. Therefore, RTB could be a good candidate to produce artificial seeds.
T. kirilowii is a medicinal plant with therapeutic properties belonging to the Cucurbitaceae family, some accessions of which exhibit a high resistance to many phytopathogens, viruses, and insect attacks [20][21][22] . Therefore, T. kirilowii can be used not only as medicinal material, but also serve as a resource for disease resistance genes for improving important Cucurbitaceae crops such as cucumber, bitter gourd, watermelon, and pumpkin. The regeneration system via SE by RTBs will promote the establishment of transformation systems in T. kirilowii, which will help increase the content of TCS and other valuable metabolites. The developed system can be used to obtain pathogen-free T. kirilowii plants because the vascular tissue of somatic embryos is independent from that of the parental explants.

Methods
Plant materials and explant preparation. T. kirilowii stems with axillary buds were treated with 75% (v/v) ethanol for 30 s, rinsed three times with sterilized distilled water, soaked in 0.1% (v/v) mercury bichloride for 8-10 min, and rinsed five times with sterilized distilled water. The sterilized stem segments were inserted in Murashige and Skoog (MS) medium 31 supplemented with 0.1 mg/L gibberellic acid (GA 3 ), 30 mg/L sucrose, and 7.8 g/L agar (pH 5.8) to obtain axillary shoots.
When the axillary shoots were approximately 1-2 cm long, they were separated and transplanted onto MS medium and cultivated at 25uC with a 16 h photoperiod (180 mmol?m 22 ?s 21 ). After 2-3 weeks of cultivation, the shoots developed into plantlets with roots. Then, the leaves or leaf discs of about 1 cm 2 in area and cut root and stem segments of about 1 cm length (for stem segments, axillary buds were avoided) were excised from the plantlets as explants for the induction of rhizoids. Histochemical and histological analyses of rhizoids and RTBs. To confirm the presence of embryonic cells in RTBs, double staining with acetocarmine and Evans blue 23,32 was used to distinguish embryonic tissue from calluses using images taken with a digital camera (EOS 600D, Canon Inc., Japan) in which embryogenic cells were stained bright red and non-embryogenic calluses were stained dark blue.
Staining with 49,6-diamidino-2-phenylindole (DAPI) was used to detect the nuclei of embryonic and callus cells, following a previously published method 23,32 . A thin slice of RTB tissue was placed on a slide and photographed with dark-field illumination using a digital fluorescence microscope (BX 61, Olympus Corporation, Japan). Cell outlines of rhizoids and RTBs were observed using borax-toluidine blue staining, according to our published protocol 32 , and images were taken using an optical microscope (BX 41, Olympus Corporation, Japan).
The microscopic frozen sections of rhizoids and RTBs at different developmental stages were created following a previously published method 23 , and imaged using an optical microscope (BX 41, Olympus Corporation, Japan).
Analyses of the internal structures of RTBs with single staining of free-hand slices. The free-hand slices (about 1 mm in thickness) cut from RTBs were put in the water and imaged using a stereomicroscope (SMZ800, Nikon Corporation, Japan). For single staining, the cut slices were immersed in staining solution (1% borax-toluidine blue dissolved in 1% sodium tetraborate as solvent) for 5 s, and rinsed with distilled water to remove residue dye. Then, the stained slices were imaged under light-field conditions using a stereomicroscope (SMZ800, Nikon Corporation, Japan).
Plantlet formation from RTBs. Two approaches were employed to form plantlets from RTBs. In the first approach, in vivo RTBs were kept on the same MS medium (supplemented with 20 mg/L TDZ) as that for the induction of RTBs to spontaneously form plantlets. In the second approach, in vitro RTBs were placed on MS medium (pH 5.   When plantlets grew to 1-2 cm, long they were separated and transferred to the rooting medium for root formation. To evaluate the frequency of regenerated plantlets from RTBs, 30 replicates of every 10 RTBs were set. DAPI staining and microscopic observation. Based on a previously published approach 32 , DAPI staining was conducted to visualize cell nuclei in images taken using a digital fluorescence microscope (BX 61, Olympus Corporation, Japan) with a mirror unit (U-MNU2), a dichroic mirror (DM400), an excitation filter (BP360), and a barrier filter (BA420).
Statistical analysis. The digital data was analysed using analysis of variance (ANOVA) using SPSS 10.0, with 99% and 95% confidence intervals.