• Journal of Plant Biotechnology
• /
• v.32 no.2
• /
• pp.135-138
• /
• 2005

Embryogenic callus was obtained from cotyledonary explants of Codonopsis lanceloata on Murashige & Skoog's medium supplemented with 1 mg/L 2,4-D. Suspension cultures of the embryogenic calli were grown on a shaker at 100 strokes/min, and then the calli were subcultured for 2 weeks in 2,4-D-free medium to produce somatic embryo. In somatic embryos at the globular stage, cotyledon initials began to differentiate themselves in the near distal end of the procambial strand. Dicotyledons, tricotyledon, tetracotyledon and fused cotyledon were differentiated from the distal ends of two, three, four and circular procambial strands, respectively. Nearly circular procambial strand in lower hypocotyls were independently differentiated into two, three, four procambial tissues at cotyledonary node and cotyledons to form somatic embryos with dicotyledon, tricotyledon, tetracotyledon. If the distal subepidermal cells of globular embryo exclusively became cotyledon initials, the torpedo or cotyledonary embryo was characterized by somatic embryos with fused cotyledon.

• Journal of Plant Biotechnology
• /
• v.40 no.1
• /
• pp.55-58
• /
• 2013

Immature embryos of Glycine max L. was cultured on Murashige and Skoog's (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). After 6 to 8 weeks of culture, immature embryos produced somatic embryos. Of somatic embryos, two cotyledonary embryo (14%), one cotyledonary embryo (37%), fused cotyledonary embryo (43%), and stunted globular embryos (6%) were observed. The procambial strand of cotyledons originated from circular procambial tissues of lower hypocotyl. The circular procambial tissues were independently divided into one or two procambial strand at the edge of cotyledonary-node, and then connected to each cotyledon to form somatic embryos with one or two cotyledons. When cotyledon was a fused type, the circular procambial strand in lower hypocotyl was continuously connected to the cotyledon. Also, somatic embryos with two cotyledons developed a functional shoot apex with the tunica-corpus structure. In contrast, somatic embryos with one or fused cotyledon formed an abnormal shoot apex without the tunica-corpus structure or with non-dome shape in the inter-cotyledonary area. These results indicated that the variation of cotyledon in somatic embryos is closely related to procambial differentiation and shoot apical meristem development.

• Korean Journal of Plant Resources
• /
• v.26 no.4
• /
• pp.511-515
• /
• 2013

Hypocotyls explants of melon seedling were cultured on Murashige and Skoog's (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) and 0.5 mg/L benzyl aminopurine (BA) for 6 weeks to produce somatic embryos. In somatic embryos produced through intervening bright yellow friable (BYF) from the explants, somatic embryos with two-cotyledon (26%) and horn-type cotyledon (74%) were observed. The procambial strand of cotyledons was originated from circular procambial tissues of lower hypocotyls. The circular procambial independently divided into two procambial strand at the edge of cotyledonary-node, and then connected to each cotyledon to form somatic embryos with two-cotyledon. When cotyledon was horn-type, the circular procambial strand in lower hypocotyls would continuously remain connected to the cotyledon. However, somatic embryos with two or horn type cotyledon formed an abnormal shoot apex without the tunica-corpus structure or dome shape in the inter-cotyledonary area. These results demonstrated that the variation of cotyledon in somatic embryos was closely related to procambial tissue differentiation and shoot apical formation.

• Journal of Plant Biology
• /
• v.39 no.2
• /
• pp.123-126
• /
• 1996

The pattern of seed storage protein, vicilin, deposition and site of intracellular localization was examined in cotyledon cells of pea (Pisum sativum) seed using the immunocytochemical methods. The vicilin was confined to the cisternae fo the rough endoplasmic reticulum and dictyosome as well as protein granules newly formed in rough endoplasmic reticulum. Vacuolar protein deposites and protein bodies were also labelled by gold particles. After small protein bodies were formed in the rough endoplasmic reticulum, they were transported to large protein bodies and then fused together. Electron dense protein granule, elaborated in the dictyosome, appears to be transported from dictyosome to protein body. A few unlabelled protein granules seem to be accumulated in other type of proteins than vicilin.

• Korean Journal of Plant Tissue Culture
• /
• v.22 no.6
• /
• pp.311-316
• /
• 1995

In an attempt to obtain intersubgeneric somatic hybrids of Pelargonium aridum and P.zonale, protoplast isolated from the two species were fused by using polyethylene glycol(PEG) and electorfusion methods. Protoplast were isolated from cotyledon and leaf tissues using MS medium containing 550 mM sucrose, 0.7% cellulase (Onozuka R-10) and 0.4% Macerozyme. The optimum number of protoplasts per mL of culture medium was 6 x 10$^4$. Protoplast fused by the electrofusion method were more active than by PEG method. Heterokaryotically fused protoplasts formed calli when cultured in MS medium containing 550 mM glucose, 1 to 2 mg/L NAA and 0.5 to 1 mg/L BA.

• Applied Microscopy
• /
• v.41 no.1
• /
• pp.61-67
• /
• 2011

The purpose of this study is to investigate the hemp (Cannabis sativa cv. Chungsam) seed structure and ultrastructure of food reserves by scanning and transmission electron microscopy. We examined the seed coat and embryo consisting of a hypocotyl-radicle axis and two cotyledons. The seed coat consisted of exotesta and endotesta. The exotesta was a mechanical layer with lignified and elongated cells, while endotesta of the underlying layers of the exotesta was consisted of two separated cell layers. The collapsed outer layer of endotesta showed the unique reticulate structures. In cotyledon cells, protein and lipid bodies occupied most of cytoplasm. Protein bodies varied in diameter from 1.8 to $5.0{\mu}m$ and possessed a protein matrix containing electron-dense globoid crystals. Numerous lipid bodies ranged from 0.8 to $3.0{\mu}m$ in diameter were distributed around the protein bodies. During the early stages of breakdown, protein bodies rapidly changed their shape into the granular feature, however, lipid bodies were gradually degradated and fused each other. The degeneration process of protein bodies and lipid bodies of cotyledon cells might be correlated with the reports which hemp seeds rapidly lose their ability to germinate.

• Journal of Plant Biotechnology
• /
• v.6 no.2
• /
• pp.91-96
• /
• 2004

The use of liquid-medium-based procedure relative to the solid media led to a 4.5-fold increase in the number of cotyledon-stage embryos. The most efficient system for multiplication and regeneration of somatic embryos was CP6 procedure with the media MSD40/MSD20/MSM6AC/FNL0S3S3GM. However, the rate of regeneration was lower. About 71％ of the embryos with dicotyledon were continued to develop the roots after desiccation treatment and 92％ of the germinated embryos produced shoots in 10 days. Of the four morphologically different types of embryos, dicotyledonous ones showed a high frequency of conversion, while only a few with fused and horn type cotyledon developed shoots. Mature somatic embryos were desiccated in empty petri dishes for 12-72 h. Embryo survival rate was the highest after 12 h of desiccation, but maximal germination was observed at 24 h. After desiccation, they were placed on MS medium without growth regulators for germination. Germinating embryos were transferred to small pots with vermiculite for plant regeneration. The etiolating the plants during the growth was resolved to add 1％ activated charcoal on hormone-free MS medium.

• Journal of Plant Biotechnology
• /
• v.2 no.1
• /
• pp.43-49
• /
• 2000

An efficient and reliable Agrobacterium transformation procedure based on TDZ (thidiazuron)-induced organogenesis was established and applied to six Brazilian eggp1ant varieties. Optimum transgenic plants recovery was achieved upon the study of the following parameters affecting transformation efficiency, using F-100 variety as a model: i) explant source; ii) pre-culture period; iii) physical state of the pre-culture medium and iv) coculture conditions. The highest frequency of kanamycin-resistant calli derived from leaf explants (5%) was obtained without a pre-culture period and co-cultivation for 24 h in liquid medium followed by five days on solid RM (regeneration medium). For cotyledon explants, best results were achieved upon a pre-culture of 24 h in liquid RM and a co-cultivation period of 24 h in liquid RM followed by three days in solid RM, resulting in a transformation Sequency of 22.7%. Kanamycin-resistant organogenic calli were also obtained from cultivars Emb, Preta Comprida, Round nose Shaded, Campineira and Florida Market. The expression pattern of an epidermis-specific promoter was studied using transformants expressing a chimaeric construct comprised by the promoter Atgrp-5 transcriptionally fused to the coding region of the gus gene. The expression pattern was similar to that previously observed in tobacco and Arabidopsis thaliana, with preferential expression at the epidermis and the stem phloem. These results support the idea that the Atgrp-5 promoter can be used to drive defense genes in these tissues, which are sites of pathogen interaction and spread, in programs for the genetic improvement of eggplant.

• Applied Microscopy
• /
• v.9 no.1
• /
• pp.57-69
• /
• 1979

The ultrastructural changes of embryo and endosperm cells were observed during the green fruit with embryo about $250{\mu}$ long to germination. 1. In the embryo cells of green fruit with embryo about $250{\mu}$ long, mitochondrial cristae and plastid are undifferentiated and dictyosome are occasionally observed. There are electron-opaque globoids in the vacuole and a lot of spherosomes in the outer layer of smooth endoplasmic reticulum. Endosperm is filled with spherosomes and electron-opaque protein bodies surrounded by spherosomes, and due to these, other organelle are not observed. 2. In the embryo cells of seeds with red seed coat, mitochondrial cristae are well developed, electron-opaque globoids increased, and vacuoles are enlarged. In the endosperm, however, spherosomes increased, protein bodies are enlarged, and electron-opaque globoidal crystals are dispersed within them. 3. In the procambium and epicotyl cells of dehiscent seed, Golgi vacuoles and vesicles are well developed, and mitochondrial cristae are also well differentiated. Spherosomes are numerously present and radicle cells, peripheral cells of hypocotyl, and vacuoles of cotyledon are well differentiated. Endosperm is filled with spherosomes containing electron-opaque granules and protein bodies are surrounded by a single membrane. There are acid phosphatase around globoids and spherosomes. 4. At the time of seeding, spherosomes markedly increased in the outer layer of cotyledon and protein bodies are also observed. Cell organelles are differentiated and plastids containing starch are also present. 5. In the outer $2{\sim}3$ layers of cotyledons, radicle cells, and peripheral cells of hypocotyl during post-seeding to germination, spherosomes and plastids with starch increased, and mitochondria and microbodies are also found around the nucleus of embryo cells. With approaching, the germination stage, in the endosperm contacting with embryo, vacuoles are well differentiated but spherosomes decreased. There increased electron-opaque materials within vacuoles. In other endosperm, with the decrease of spherosome, mitochondria increased and electro n-opaque globular bodies are formed and gradually increased. The outer layer of protein bodies are reduced while electron-transparent portions are enlarged and fused together to occupy the outer layer where small particles are formed. 6. In the endosperm of germination stage, spherosomes decreased while protein bodies, are fused together to form 2 or 3 within a cell.