In general, somatic embryos generated through plant tissue culture are known to be functionally and morphologically similar to natural zygotic embryos, but there is a significant differences (Ammirato 1987). Some studies have reported differences in the cotyledon number and structure of somatic embryos (Choi et al. 2005; Lee and Soh 1993), and the causes are the type and concentration of carbohydrates (Choi 2020; Soh et al. 2001), 2,4-D (Choi et al. 1994), ABA and BAP (Lee and Soh 1993), auxin (Liu et al. 1993) added to the medium, etc. have been reported. In addition, since abnormality of somatic embryos generated in vitro cultures show a low rate of plant regeneration (Choi and Kwon 2013a; Gray and Mortensen 1987; Soh et al. 2001), it is firstly necessary to understand the process of development of somatic embryos in order to conduct research on the mass production or gene introduction using somatic embryos.

It was reported the theoretical background that cotyledon formation in somatic embryo differentiates from initial cells of lower epidermis at the tip of globular embryo (Ammirato 1987), and also reported a study that differentiated into cotyledonous nodes and cotyledons after the differentiation a procambial tissue from epicotyl or hypocotyl regions of somatic embryos (Choi et al. 2005; Choi and Kwon 2013a, 2013b; Lee 1993). On the other hand, somatic embryos having abnormal cotyledons formed from in vitro cultures were reported that reduce the germination rate by delaying or inhibiting the conversion to plants by abnormal development of apical meristem (Choi et al. 2005; Choi and Kwon 2013a, 2013b; Soh et al. 2001), or there is also another that abnormal cotyledon differentiation of somatic embryos results in the development of multiple shoots from abnormal shoot meristem (Lee 1993). Like these, since the cotyledon formation of somatic embryos is presumed to be closely related to the differentiation of procambium tissue, it is necessary to understand for the cotyledon differentiation of somatic embryos.

The purpose of this study is to report the histological observations on the relationship between the differentiation of the procambium tissue and the cotyledon development in the early stage of cotyledon formation of normal and abnormal somatic embryos generated in-vitro cultures of carrots.

Plant material

Carrot seed (Daucus carota L., cv. Sin Heuk Jeon 5 Chon), which is purchased from Asia Seed Korea was used in this study.

Formation of somatic embryos

Carrot seeds were immersed in 70% alcohol for 1 min and 1% sodium hypochlorite solution for 15 min for surface sterilization, and then washed 3-5 times with sterilized water. 15~20 of the sterilized seeds were cultured in each Petri dish containing MS basal medium (Murashige and Skoog 1962) for germination. After 2 weeks of culture, sterilized-seedlings were obtained, and then embryonic callus from hypocotyl explants of germinated seedling was induced while culturing in MS medium with 1.0 /mg/L 2,4-D for 5~6 weeks. Embryogenic callus induced from hypocotyl explants were carefully selected under a dissecting microscope, and then proliferated by subculture in the same medium at intervals of 4 weeks. Somatic embryogenesis from the embryogenic callus was performed by liquid culture and all methods were performed according to Lee et al.’s method (2023). Somatic embryos formed from liquid cultures were classified according to cotyledon number and morphology under a dissecting microscope. Somatic embryos with two, three and four cotyledons were used as materials for histological analysis.

Analysis of somatic embryos by scanning electron microscope (SEM)

The cotyledon development in somatic embryos was analysed with a SEM. Somatic embryos with heart to torpedo stages were fixed in 2.5% glutaraldehyde solutions at 4°C for 4 hours and then dehydrated in an alcohol series. After drying with a critical point dryer for 2 hours or more, the material was fixed on the Stab using double-sided tape and then silver pest was applied to the attached sample, and a 100Å-thick gold skin was applied with an ions evaporator and observed with a scanning electron microscope.

Histological observation

In order to histologically observe the development of cotyledons and the differentiation of the procambial tissue in carrot somatic embryos, somatic embryos with two, three and four cotyledons formed from in vitro culture were carefully selected under a dissecting microscope (Lee et al. 2023). After fixing in FAA for more than 24 hours, it was dehydrated with butanol series and embedded in paraplast. 10 µm-thick sections were sequentially cut using a rotating microtome, stained with hematoxylin, and then observed under a light microscope according to the method of Choi and Kwon (2013a).

After 2 weeks of cultures, callus began to form from cutting edges of the hypocotyl explant on the medium with 1.0 mg/L 2,4-D, and a friable yellowish callus was newly proliferated with somatic embryos that was first appeared at the 4th week. Proliferation of embryogenic competent cells or cell mass was obtained by culturing in MS liquid medium supplemented with 1.0 mg/L 2,4-D every 2 weeks for 6 weeks (Fig. 1A), and then a large amount of somatic embryos at globular or heart stages could be obtained when the cells or clumps of somatic embryogenic competent were cultured in MS liquid medium without hormone for two weeks (Fig. 1B, C, D, E). Somatic embryos derived from liquid culture could be classified into normal somatic embryos with two cotyledons (Fig. 1F) and abnormal somatic embryos with three or four cotyledons under a dissecting microscope and scanning electron microscope (Fig. 1G). In general, it has been reported that somatic embryogenesis of plants occurs in a medium containing with 2,4-D (Buchheim et al. 1989; Garcia et al. 2019). In particular, it has been known that the optimal concentrations for somatic embryogenesis were 1.0 mg/L 2,4-D in case of carrot (Lee et al. 2023) and 0.1-4.0 mg/L 2,4-D ranges in case of immature embryo of soybean (Choi et al. 1994). Like these, it can be seen that 2,4-D has a very important effect on the somatic embryogenesis in many plants (Garcia et al. 2019; Raghavan 2004). Also, it is known two views, that the development of cotyledon in somatic embryos is achieved by division of cotyledon initial cells at the tip of the somatic embryo at the globular stage (Bhojwani and Arumugam 1993), or that a somatic embryo with two cotyledons is formed when two primodiums are formed, a somatic embryo with three cotyledons when three primodiums are formed, and a somatic embryos with fused cotyledon are formed when cylindrical division occurs after an circular meristem is formed at the tip of the globular stage (Ammirato 1987). In this study, it was observed that a somatic embryo with two or three cotyledons was formed when two or three primodia were formed in the circular ring structure at the tip of the globular stage of somatic embryo, respectively (Fig. 1F, G). These findings showed similar trends to those of Ammirato (1987) as mentioned above.

Figure 1. Somatic embryogenesis and cotyledon variations of somatic embryos obtained from cell cultures of Daucus carota L. embryogenic callus cultured in liquid medium without hormone for two weeks. A: Typical embryogenic cell; B: somatic embryos in globular stage; C: somatic embryos in heart stage; D, E: two cotyledon primordia at the tip region of heart- or torpedo-shaped somatic embryos; F: somatic embryo containing two cotyledons; G: somatic embryo containing three cotyledons

In histological observation of somatic embryos with two, three and four cotyledons, the procambial tissue of somatic embryos with two cotyledons had a circular shape in the hypocotyl region, and this circular strand of procambium was divided into two independent tissue toward the cotyledonary node, and differentiated into each cotyledon (Fig. 2). In addition, the circular procambial strand in hypocotyl region of somatic embryos with three or four cotyledons was divided into three or four independent tissue into the cotyledonary node and each cotyledon, respectively (Fig. 3, 4). Likewise, the circular procambium tissue was differentiated in the hypocotyl region of the normal somatic embryo with two cotyledons and the abnormal somatic embryo with three or four cotyledons, but it was found that the differentiation of the procambium tissue was different in the cotyledonary node and the cotyledon region. In general, it is known that the main causes of abnormal somatic embryos are hormones and carbon sources added to the medium (Garcia et al. 2019; Kageyama et al. 1990), and this phenomenon is reported to be caused by the polar shift of endogenous auxin in the embryo at the globular stage (Liu et al. 1993). Also, it is known that the variation of cotyledon number are closely related to the differentiation of the procambium tissue in the heart stage of somatic embryo (Choi et al. 2005; Raghavan 2004). In this study, the variation of cotyledon number in the somatic embryos also showed that the circular procambium strand showed in the hypocotyl region was differentiated in the polar direction of cotyledon and radicle, and the procambium differentiation was found that was closely related to the cotyledon development of the somatic embryos. In this way, the somatic embryos generated during the in vitro culture can be not only normal, but also somatic embryos with abnormal cotyledons can be generated, and this is believed to be related to the development of procambium when developing from globular stage somatic embryos to heart stage somatic embryos.

Figure 2. Cross-sections of a somatic embryo containing two cotyledons in cell cultures of Daucus carota L. Two procambial tissues at the region of cotyledon (A) and cotyledonary node (B) are shown. C: Transformation into two procambial tissues in the upper hypocotyl (arrows, C). D: Nearly circular procambial tissue in the lower hypocotyl. All bars = 75 µm

Figure 3. Cross-sections of a somatic embryo containing three cotyledons in cell cultures of Daucus carota L. A, B: Three procambial tissues at the region of cotyledon. C: Transformation into three procambial tissues in the upper hypocotyl (arrows, C). D: Nearly circular procambial tissue in the lower hypocotyl. All bars = 70 µm

Figure 4. Cross-sections of a somatic embryo containing four cotyledons in cell cultures of Daucus carota L. A, B: Four procambial tissues at the region of cotyledon. C: Transformation into four procambial tissues in the upper hypocotyl (arrows, C). D: Nearly circular procambial tissue in the lower hypocotyl. All bars = 70 µm

This study was supported by research funds from Nambu University, 2023.