Medicinal plants are valuable sources of medicinal and many other pharmaceutical products. The conventional propagation method is the principal means of propagation and takes a long time for multiplication because of a low rate of fruit set, and/or poor germination and also sometimes clonal uniformity is not maintained through seeds. The plants used in the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increase in the demand for the crude drugs, the plants are being overexploited, threatening the survival of many rare species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. The purpose of the present review is to focus the application of tissue culture technology for in vitro propagation via somatic embryogenesis and organogenesis and the cell suspension culture with suitable examples reported earlier. An overview of tissue culture studies on important Chinese medicinal plants and related species is presented.

Plant proteins, when used as dietary protein, are generally incomplete in nutrition due to their deficiency in several essential amino acids, for example, lysine and tryptophan in cereals and methionine and cysteine in legumes. Attempts to breed crops with increased levels of lysine and methionine have been less than satisfactory. Modern biotechnology offers alternative approaches for rectifying this nutrition deficiency. In the past decade, several transgenic strategies aimed at modifying the amino acid composition of plant proteins and enhancing the content of specific essential amino acid(s) for nutrition improvement have been developed and tested. These include synthetic proteins, modification of protein sequences, over-expression of heterologous or homologous proteins, and metabolic engineering of the free essential amino acid pool and protein sink. The progress and potential of these approaches and studies are reviewed. As plant proteins are the primary source of all dietary protein consumed by humans and animals and are inexpensive to produce in comparison with meat, improving their quality will make a significant contribution to our future food needs. The research and development in this area of interest is making promising progress towards this endeavor.

Among the established plant cell lines, tobacco BY-2 cell line is unique, as it can be highly synchronized by established procedures. Because of this reason, the cell line has become invaluable for various studies, particularly on cell cycle issues. The importance of several characteristics of the cell line, some of which have not been handled thus far, is described in this article. We also include some preliminary characterization of an auxin-autotrophic cell line 2B-13 derived from the BY-2 cell line. Thus, the repertoire using the BY-2 cell line in plant sciences is expanding. The importance of this cell line could increase further as the expressed sequence tag (EST) database of the cell line became publicly available recently. The scientific achievements on this cell line that have been accumulated in recent years are being compiled and will be published as the 53rd volume in the monograph series of Biotechnology in Agriculture and Forestry (Nagata et al., 2004); readers are referred to this source.

A protocol has been developed for in vitro plant regeneration from cotyledonary nodes of Pterocarpus marsupium Roxb. Multiple shoots were induced from cotyledonary nodes derived from 20-d-old axenic seedlings grown on Murashige and Skoog (MS) medium containing 2.22–13.32 μM benzyladenine (BA) or 2.32–13.93 μM kinetin alone or in combination with 0.26 μM α-naphthaleneacetic acid (NAA). The highest frequency for shoot regeneration (85%) and maximum number of shoots per explant (9.5) were obtained on the medium supplemented with 4.44 μM BA and 0.26 μM NAA after 15 wk of culture. A proliferating shoot culture was established by repeatedly subculturing the original cotyledonary nodal explant on fresh medium after each harvest of the newly formed shoots. Nearly 30% of the shoots formed roots after being transferred to half-strength MS medium containing 9.84 μM indole-3-butyric acid (IBA) after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on a peat moss, soil, and compost mixture (1:1:1). About 52% of plantlets were successfully acclimatized and established in pots.

An efficient regeneration protocol has been developed from leaf explants of Pluchea lanceolata, a medicinally important plant of the family Asteraceae. Nodular callus was initiated from young leaf segments cultured on Wood and Braun medium containing 2.0% sucrose (WB) supplemented with kinetin. On WB 5.0 mg l^−l kinetin, 100% plant regeneration with 14 ± 0.5 plantlets was obtained. Regenerated plantlets with well-developed root systems were transferred to pots and subsequently to the field. With respect to the effect of metals on morphogenic events and quercetin content of the cultures, lower concentrations of CuSO₄ proved to be beneficial but higher concentrations were detrimental. The presence of 200 μM ZnSO₄ or 150 μM CuSO₄ also resulted in the highest level of quercetin in regenerants. The yield of quercetin in culture varied with stages of differentiation.

The diploid cotton species can constitute a valuable gene pool for the more agronomically desirable cultivated tetraploid cultivars and offer better opportunities to study gene structure and function through gene knockouts. In order to exploit these advantages, a regeneration system is required to achieve these transformation-based goals. Carbohydrate source and concentration were evaluated to improve somatic embryo (SE) production and desiccation treatments to improve the conversion efficiency of SEs to plants in a diploid Gossypium arboreum accession, A₂-9 (PI-529712). Improved SE numbers and their subsequent conversion into plantlets was achieved with a Murashige and Skoog (MS)/sucrose-based medium M₂ [0.04 M sucrose, 0.3 μM α-naphthaleneacetic acid (NAA)]. On this medium, 219 embryos per g initiated, and close to 11% of these embryos germinated into plantlets. Neither a 5-d desiccation treatment of embryogenic callus previously cultured in liquid medium nor filter paper insertion improved the numbers of SEs induced or their conversion to plantlets. A 3-d desiccation period resulted in improved plant regeneration. When immature G. arboreum SEs induced on M₁ (0.2 M glucose, 2.6 μM NAA, and 0.2 μM kinetin) medium underwent a 3-d desiccation treatment, 49% of these immature SEs were converted to plantlets after a 4-wk period on M₂ medium. These improved results will help to pave the way for future genetic transformation and associated gene structure and function studies utilizing G. arboreum. These results, in particular the 3-d desiccation treatment, can also be incorporated into regeneration protocols to improve the regeneration efficiency of other Gossypium species.

Excised cotyledons from 8-d-old pumpkin (Cucurbita pepo L.) seedlings were inoculated with Agrobacterium rhizogenes and cultured on hormone-free Murashige and Skoog medium. At the site of inoculation, transformed hairy roots were successfully induced by using wild strains 8196 (mannopine-type) and 15834 (agropine-type). After a subsequent transfer on a solid MS medium without hormones, roots obtained by transformation with strain 15834 failed to form stable hairy root cultures, while several hairy root lines were established with strain 8196. Three hairy root lines, Cp1, Cp2, and Cp31, have spontaneously generated callus with embryo-like structures after more than 3 yr of growth on the solid medium. The callus proliferation was more frequent when the autoclaving of nutrient medium, pH 5.7, was prolonged to 30 min. Separated calluses continued to proliferate and generated embryos with abnormal morphology. The combination of indole-3-acetic acid and benzyladenine had a favorable influence on embryogenesis and organogenesis in the Cp31 callus line. The Southern analysis of Cp31 root and embryo DNA confirmed the presence of the T-DNA of Agrobacterium rhizogenes.

Eggplant (Solanum melongena L.) was efficiently regenerated from cultured roots of 15-d-old seedlings on Murashige and Skoog (MS) medium containing 0.45 μM thidiazuron and 13.3 μM 6-benzyladenine. Within 28 d of culture initiation, induction of organogenic calluses and subsequent differentiation into shoot buds were observed. Shoot buds upon subculture to MS basal medium elongated into healthy shoots. Excised shoots (2–4 cm) were rooted on Soilrite^® irrigated with water either in vitro or in vivo. Plants with well-developed root systems were established under field conditions after hardening in the glasshouse, where they developed into flowering plants and produced mature fruits with viable seeds.

A protocol for in vitro propagation using direct induction of shoot buds from leaf explants of in vitro-raised shoots of Rosa damascena var. Jwala is reported. The present study is the first report on direct shoot regeneration in scented roses. Elite plants raised from nodal explants and maintained for over 2 yr in vitro on a static liquid shoot multiplication Murashige and Skoog (MS) medium supplemented with 5.0 μM benzyladenine (BA) and 3% sucrose were used. Petioles from fully developed young leaves, obtained after 4 wk of pruning of old shoots, were found to be ideal for regeneration of shoots. Initially the explants were cultured in an induction medium [half-strength MS 3%sucrose 6.8 μM thidiazuron 0.27 μM α-naphthaleneacetic acid (NAA) 17.7 μM AgNO₃] and subsequently transferred to the regeneration medium (MS 2.25 μM BA 0.054 μM NAA) after 7, 14, 21, 28, and 35 d. The highest shoot regeneration response (69%) was recorded when shoots were kept in the induction medium for 21 d and later transferred to regeneration medium. Histological studies revealed direct formation of shoot buds without the intervening callus phase. In vitro rooting of micro-shoots was accomplished within 2 wk on half-strength MS liquid medium supplemented with 10.0 μM IBA and 3% sucrose for 1 wk in the dark and later transferred to hormone-free medium and kept in the light. Plantlets, remaining in the latter medium for 5–6 wk when transferred to soil, showed 90% survival.

For the first time, regenerated plantlets were obtained from immature zygotic embryos of mango (Mangifera indica L.) through direct somatic embryogenesis. Pro-embryogenic mass (PEM)-like structures, which are differentiated as clusters of globular structures, were easily induced directly from the abaxial side of cotyledons from immature fruits, 2.0–3.5 cm diameter by a 2-wk culture period on a modified Murashige and Skoog medium with 5 mg l⁻¹ (25 μM) indole-3-butyric acid (IBA). Conversion of somatic embryos into plantlets was achieved after 4 wk of culture on the conversion medium containing 5 mg l⁻¹ (23 μM) kinetin. Secondary somatic embryogenesis could also be obtained directly from the hypocotyls of mature primary somatic embryos cultured on the conversion medium. In our experimental system, only minor problems were noted with browning of cultures.

A highly reproducible method for regeneration of Coffea arabica and C. canephora plants via direct somatic embryogenesis from cultured leaf and stem segments of regenerated plants was developed. Embryogenesis was influenced by the presence of triacontanol (TRIA) in the medium. TRIA incorporated at 4.55 and 11.38 μM in half-strength MS basal medium containing 1.1 μM 6-benzyladenine (BA) and 2.28 μM indole-3-acetic acid (IAA) induced direct somatic embryogenesis in both species. A maximum of 260 ± 31.8 and 59.2 ± 12.8 somatic embryos per culture were induced from in vitro leaf explants of C. arabica and C. canephora, respectively. TRIA also induced embryo formation from in vitro stem segment callus tissues along with multiplication of primary embryos into secondary embryos. By using TRIA, it was possible to obtain somatic embryogenesis in C. arabica and C. canephora.

In vitro propagation of Andrographis paniculata (Burm. f.) Wallich ex Nees through somatic embryogenesis, and influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on induction, maturation, and conversion of somatic embryos were investigated. The concentration of 2,4-D in callus induction medium determined the induction, efficacy of somatic embryogenesis, embryo maturation, and conversion. Friable callus initiated from leaf and internode explants grown on Murashige and Skoog (MS) medium supplemented with 2.26, 4.52, 6.78, and 9.05 μM 2,4-D started to form embryos at 135, 105, 150, and 185 d, respectively, after explant establishment. Callus initiated at 13.56 μM 2,4-D did not induce embryos even after 240 d, whereas those initiated on MS medium with 4.52 μM 2,4-D was most favorable for the formation and maturation of somatic embryos. Callus subcultured on the medium with reduced concentration of 2,4-D (2.26 μM) became embryogenic. This embryogenic callus gave rise to the highest number of embryos (mean of 312 embryos) after being transferred to half-strength MS basal liquid medium. The embryos were grown only up to the torpedo stage. A higher frequency of embryos developed from callus initiated on 2.26 or 4.52 μM 2,4-D underwent maturation compared to that initiated on higher concentrations of 2,4-D. The addition of 11.7 μM silver nitrate to half-strength MS liquid medium resulted in 71% of embryos undergoing maturation, while 83% of embryos developed into plantlets after being transferred to agar medium with 0.44 μMN⁶-benzyladenine and 1.44 μM gibberellic acid. Most plantlets (88%) survived under field conditions and were morphologically identical to the parent plant.

A micropropagation procedure for the adult cherimoya tree (Annona cherimola Mill.) is described. Axillary shoot proliferation was obtained after culturing nodal sections from Annona cherimola cv. ‘Fino de Jete’, on Murashige and Skoog (MS) medium supplemented with 2.28 μM zeatin. Roots were induced after preincubation of shoots for 3 d in light on MS basal medium supplemented with 1 g l⁻¹ activated charcoal, followed by culturing for 10 d (7 d dark and 3 d light) on MS medium with 492 μM indole-3-butyric acid (IBA), 15 g l⁻¹ sucrose, and 200 mg l⁻¹ citric acid. Sixty-eight percent of induced shoots rooted after transferring to the same medium without auxin and with the macroelements at half strength and the sucrose at 20 g l⁻¹. About 65% of rooted shoots survived after acclimatization. The procedures described herein may prove useful for clonal micropropagation of selected genotypes of cherimoya.

The influence of light quality on competence and determination for organogenesis was investigated using lettuce cotyledon explants. Lettuce seedlings from four genotypes were germinated in the dark or under white, red, or blue light. Cotyledon explants were excised and cultured on a shoot-inducing medium for 28 d under white light. Germination in the dark reduced shoot numbers, suggesting that light improves the competence of explants for organogenesis. When explants from seedlings germinated under white light were cultured under different light qualities, blue was found to inhibit shoot production while red light either promoted production or had no effect on shoot number compared to controls. Treatment with blue plus red light failed to overcome the inhibition by blue light. To ascertain the temporal responses of explants to light quality, they were cultured under red or blue light prior to transfer to the alternate treatment. Exposure to blue light within 7 d of excision permanently reduced explant competence for organogenesis. Exposure after this time had a minimal effect. These results suggest that both phytochrome and cryptochrome can regulate shoot production from lettuce cotyledon explants and blue light can only inhibit organogenesis, in lettuce, during a relatively small developmental window.

Growth sensitivity of four local grapevine (Vitis vinifera) varieties, Ashlamesh, Helwani, Kassofee, and Khoudeiry, were evaluated for salt. They were cultured on DSD1 medium until rooting stage, then they were transferred to a liquid DSD1 medium containing 0, 10, 20, 30, 40, 80, 120, or 150 mM NaCl for 30 d. The shoot length and leaf number of Ashlamesh, Helwani, and Kassofee were significantly increased at 10 and/or 30 mM NaCl, whereas, 150 mM NaCl decreased shoot length of all varieties except Kassofee. The presence of NaCl at 80 mM or higher concentrations decreased the chlorophyll content and root number of all varieties, while 30 mM NaCl increased root number of Kassofee.

The life table of a local strain of grape phylloxera was determined to evaluate the resistance of the most commonly used rootstocks: Ru140, R99, and 3309C, and one local variety ‘Helwani’. The study was carried out by applying both in vitro dual culture and small root pieces testing systems. The results showed that there was a great variation in percentage mortality of immature stages, number of eggs laid, adult fertility, oviposition period, and developmental time between all tested rootstocks and the local variety, regardless of the applied testing systems. Based on the population prediction, Helwani would be a suitable host as it would be susceptible to the destructive insects in the field and the rootstocks would be resistant. However, Ru140 rootstock was more susceptible than R99 and 3309C rootstocks.

Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog's medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM), but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.

Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.

Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation–dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopreserved shoot tips was not affected by the period of cold acclimation of in vitro mother plants.