Somatic embryogenesis and plant regeneration

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The suggestion of somatic embryogenesis in ginseng tissue cultures was first made by Butenko and her colleagues (1968) but at that time all attempts to achieve the regeneration of whole ginseng plants from isolated embryo-like structures failed. Pursuing their work on the culture of ginseng root tissues Chang and Hsing (1978) obtained embryoids from root derived callus in defined conditions. Regeneration systems via somatic embryogenesis were established in root callus (Chang and Hsing, 1980), in zygotic embryo callus (Lee et al., 1990) and in protoplast derived callus (Arya et al., 1991). High-yield and short period embryogenesis via callus induced by the culture of young flower buds on the MS medium supplemented with 2,4 D in the dark was developed in 1988 by Shoyama et al. Later, Arya et al. (1993) reported a rapid somatic embryo formation, obtained due to secondary and tertiary embryogenesis. Embryogenic callus was initiated from immature zygotic embryos of ginseng and the somatic embryos were multiplied by adventitious embryogenesis, their growth and development being dependent on growth hormones in the medium.

Formation of embryoids has also been observed in callus cultures derived from leaf lamina after 6 month cultivation on the basal medium supplemented with NAA and kinetin (Cellarova et al., 1992). Histological analysis of embryogenic calli showed the presence of embryo like structures in various stages of development and many anomalous structures such as two embryoids of the same origin in different stages of development, meristemization of torpedo stage embryoids and asymmetrical development of cotyledons. Further development required a change of culture conditions.

Immature zygotic embryos and the cotyledonary segments of mature zygotic embryos of ginseng actively produced somatic embryos on MS basal medium without any growth regulators (Choi and Soh, 1996a, 1996b), but intact embryos, embryos with half of the cotyledons removed and excised plumules and radicles did not produce any somatic embryos. Such somatic embryos from cotyledon segments arose only near the excised portion at the cotyledonary base. The polar somatic embryogenesis from the cotyledon base was not affected irrespective of the segment sizes of the cotyledons. However, the frequency of somatic embryos formation and their growth rate were highly influenced by the size of segments. The authors concluded that the somatic embryogenesis from excised or wounded cotyledonary segments occurred by the complementary action of both wound response and tissue polarity. When a small needle prick or incision was made on the surface of the cotyledonary segments, the somatic embryos were formed near the wounded portion as well as at the basal excised portion. Somatic embryogenesis did not occur on or near the wounded or excised portion situated in the acropetal direction of cotyledon although the same wounding response was observed on those sites as well.

Embryoids isolated from the callus and subcultured either on half-strength or B5 medium supplemented with BA and GA3 develop into plantlets. Chang and Hsing (1980) showed in vitro flowering of plantlets regenerated from mature root callus. The apical meristem located between two cotyledons transformed into a peduncle terminated by a simple umbel with 3 to 15 flowers. The regeneration procedure described by Shoyama et al. (1988) using half strength

Figure 9. Shoots differentiated from embryoids of leaf origin of Panax ginseng.

MS medium with GA3 and BA was suitable also for embryoids of reproductive (Shoyama et al., 1988) and vegetative (Cellarova et al., 1992) origin (Fig. 9).

About 90 per cent of the pollen grains were fertile. Arya et al. (1991) described for the first time an efficient procedure for isolation, culture and regeneration of protoplasts into flowering plantlets through somatic embryogenesis. A 4-year old embryonic cell line of Panax ginseng capable of regeneration was used. Most plantlets produced more than two epicotyls.

However, further experiments on the genetic uniformity of plantlets produced in such a manner are needed. Asaka et al. (1993) developed a technology to induce embryoids by a moderate high temperature treatment from multiple shoots of P. ginseng. These embryoids were formed on the surface of the differentiated tissue. Normal plantlets were regenerated from the embryoids by transplanting them on hormone free medium.

Direct somatic embryogenesis from cultured primary somatic embryos capable of plant formation without intervening callus phase was obtained by Arya et al. (1993). Cotyledonary stage somatic embryos developed shoot axes when transferred to MS medium supplemented with kinetin and later roots were formed when they were transferred to MS medium with kinetin and GA3. Secondary somatic embryos also formed plantlets in a one step process on half strength MS medium supplemented with BA or kinetin and GA3.

Somatic embryogenesis induced in vitro is a process which is considered to be a biotechnological alternative to cover the demand for ginseng plants which has significantly increased over recent years. However, when somatic embryos are differentiated from callus, they may not always be able, due to numerous structural abnormalities, to germinate and form normal plantlets. This problem can be overcome when ginseng seeds are used as sources of zygotic embryos. Germinating zygotic embryos give rise to somatic embryos which are able to grow directly into plants. This procedure significantly shortens the regeneration process and provides as many as thousands of ginseng plants by culturing just one seed.

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