Cryopreservation (liquid nitrogen, −196°C) represents the only safe and cost-effective option for long-term conservation of germplasm of non-orthodox seed species, vegetatively propagated species, and of biotechnology products. Classical cryopreservation techniques, which are based on freeze-induced dehydration, are mainly employed for freezing undifferentiated cultures and apices of cold-tolerant species. New cryopreservation techniques, which are based on vitrification of internal solutes, are successfully employed with all explant types, including cell suspensions and calluses, apices, and somatic and zygotic embryos of temperate and tropical species. The development of cryopreservation protocols is significantly more advanced for vegetatively propagated species than for recalcitrant seed species. Even though its routine use is still limited, there are a growing number of examples where cryopreservation is employed on a large scale for different types of materials, including seeds with orthodox and intermediate storage behaviour, dormant buds, pollen, biotechnology products, and apices sampled from in vitro plantlets of vegetatively propagated species. Cryopreservation can also be employed for uses other than germplasm conservation, such as cryoselection, i.e., the selection through freezing of samples with special properties, or cryotherapy, i.e., the elimination of viruses from infected plants through apex cryopreservation. Because of its high potential, it is expected that cryopreservation will become more frequently employed for long-term conservation of plant genetic resources.

Over the past 20 years, DNA-based biotechnologies have been applied to agricultural production and many crops with new and useful attributes have been cultivated in various countries. The adoption of this new technology by farmers has been swift, and benefits in terms of increased production per unit land and environmental benefits are becoming obvious. In forestry, the application of biotechnology is somewhat lagging behind and to date there are no commercial plantations with genetically modified trees. However, most tree species used in plantation forestry have been genetically transformed, and results demonstrate the successful and correct expression of new genes in these plants. At the same time, this new technology is being viewed with concern, very similar to the concerns voiced over the use of genetic engineering in agriculture. This paper discusses some of the issues involved for world forestry, with particular focus on future demand for timber and timber products and how modern biotechnology can contribute to meet the growing demand. Tree genetic engineering techniques will be outlined, and results reviewed for a number of species. Concerns over the use of this new technology will be described and analyzed in relation to scientific considerations.

Genetic transformation provides the means for modifying single horticultural traits in perennial plant cultivars without altering their phenotype. This capability is particularly valuable for perennial plants and tree species in which development of new cultivars is often hampered by their long generation time, high levels of heterozygosity, nucellar embryony, etc. Most of these conditions apply to many tropical and subtropical fruit crops. Targeting specific gene traits is predicated upon the ability to regenerate elite selections of what are generally trees from cell and tissue cultures. The integrity of the clone would thereby remain unchanged except for the altered trait. This review provides an overview of the genetic transformation of perennial tropical and subtropical fruit crops, i.e., citrus (Citrus spp.), banana and plantain (Musa groups AAA, AAB, ABB, etc.), mango (Mangifera indica L.), pineapple (Ananas comosus L.), avocado (Persea americana Mill.), passion fruit (Passiflora edulis L.), longan (Dimocarpus longan Lour.), and litchi (Litchi chinensis Sonn.).

Successful fundamental or basic research, while being stimulated by applied studies, provides the development of new technologies for the benefit of mankind. Photoautotrophic micropropagation or micropropagation using sugar-free medium is no exception from this generalization. The concept of photoautotrophic micropropagation is derived from research that revealed the relatively high photosynthetic abilities of chlorophyllous cultures such as leafy explants and plantlets in vitro. To meet the ever-increasing demand for quality transplants, the scaling-up of photoautotrophic micropropagation systems, for commercialization, has become necessary. This article reviews the recent advancement in the development and utilization of large culture vessels for photoautotrophic micropropagation with special emphasis on the feasibility of the system for the commercial-scale propagation. The review also includes choices for supporting material, ventilation type, planting density, vessel volume, and vessel sterilization procedure, and problems and solutions to achieve uniform growth in a large culture vessel. A case study of the commercial application of a photoautotrophic micropropagation system using large culture vessels, which recently has been established in Kunming, China, is also presented in this article.

High-frequency somatic embryogenesis and plant regeneration were achieved on callus developed from root, stem disc, leaf, and scape explants of Eryngium foetidum L. (Apiaceae). Calluses developed on Murashige and Skoog (MS) medium fortified with 5.37–10.74 μM α-naphthaleneacetic acid (NAA) in combination with 2.32 or 4.65 μM kinetin (Kn) formed somatic embryos after transfer to liquid half-strength MS (½MS) medium with 2.69 μM NAA and 1.16 μM Kn. Furthermore, promotion of embryo formation was also observed in callus developed on medium with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and Kn after transferral to ½MS liquid medium containing 10% coconut water (CW)/coconut toddy (CT). Somatic embryos developed into plantlets at the highest frequency (98%) after transferral to solid ½MS medium containing 10% CW/CT. All the plantlets were acclimatized in soil and survived under field conditions, and they were morphologically indistinguishable from the source plant.

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 of responding explants (85%) and maximum number of shoots per explant (9.5) were obtained on MS 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 after 25 d of culture. Fifty percent of shoots were also directly rooted as microcuttings on peat moss, soil, and compost mixture (1:1:1). About 52% plantlets rooted under ex vitro conditions were successfully acclimatized and established in pots.

Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mg l⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mg l⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.

An in vitro protocol for Ficus carica cv. ‘Roxo de Valinhos’ was optimized. Nodal explants containing two buds were excised from field-grown mature plants, and transferred to different proliferation media consisting of combinations of distinct concentrations of activated charcoal with benzyladenine (BA), kinetin with gibberellic acid (GA₃), and WPM (woody plant medium) with kinetin. The regular strength of WPM in combination with 0.5 mg l⁻¹ kinetin was the best condition for shoot proliferation of Ficus carica ‘Roxo de Valinhos’ plants. The addition of activated charcoal in the medium completely inhibited shoot proliferation. The inclusion of BA in the medium induced excessive callus formation as well as small and vitrified shoots, while GA₃ induced excessive elongation associated with vitrification, chlorosis, and tip-burned shoots.

Improved in vitro tissue culture systems are needed to facilitate the application of transgene technology to the improvement of sugar beet germplasms. Several commercially important sugar beet breeding lines (SDM 3, 5, 8, 9, 10, 11, HB 526, and CMS 22003) and commercial varieties (Roberta and Gala) were tested for their regeneration capacity through adventitious shoot organogenesis from cotyledons, hypocotyls, root/hypocotyl/shoot transition zone tissues, and leaf lamina and petiole via an intervening callus phase. Callus induction and adventitious shoot regeneration was dependent on genotype and combinations of plant growth regulators. With cotyledon or hypocotyl explants, SDM 3 and 10 showed a better response on adventitious shoot regeneration in medium containing benzyladenine (BA) and 2,3,5-triiodobenzoic acid or 1-naphthaleneacetic acid (NAA) than SDM 11, 5, and 9. Shoot regeneration was obtained from hypocotyl–root or hypocotyl–shoot transition zone tissue in SDM 9, 10, and HB 526 grown on PGo medium supplemented with BA to induce callus, and the regeneration frequency was 25%. Adventitious shoots were also regenerated from leaf explants of SDM 3 and 9 cultured on medium containing NAA for callus induction and BA and NAA to induce shoot regeneration, and in SDM 10 and CMS 22003 cultured on medium containing BA for callus induction and to induce shoot regeneration.

Micropropagation of Scabiosa caucasica cv. Caucasica Blue was achieved by culturing, separating axillary and adventitious shoots, or node sectioning on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA). The highest frequency of adventitious shoots regenerated from nodal or internodal explants and leaf blade (with or without petiole) appeared to occur on MS medium with 4.4 and 18 μM BA, respectively. Addition of 0.19 or 1.9 μM α-naphthaleneacetic acid to the BA-containing medium promoted callus formation and reduced shoot organogenesis. During micropropagation, shoot nodal explants derived from in vitro shoots cultured on MS medium supplemented with 4.4 μM BA yielded 8.9 shoots per explant within 40 d after culture initiation.

With the objective of using microtubers for conservation of potato germplasm, the main effects of genotype, abscisic acid (ABA), and sucrose level, and of their interactions on biomass production, microtuberization, microtuber dormancy, and dry matter content, were studied. ABA decreased both microtuber production and microtuber dormancy, whereas higher concentrations (60–80 g l⁻¹) of sucrose promoted biomass production, microtuber production as well as microtuber dry matter content. Microtubers stored under diffused light had longer dormancy than those kept continuously in the dark. Interactions among various factors conditioned the main effects for some characters. In vitro performance of the genotypes studied was related to their known performance under in vivo conditions for most of the characters. Microtubers produced on media devoid of ABA and containing high sucrose concentrations and N⁶-benzyladenine (44.38 μM) could be stored for 12 mo. under diffused light at 6 ± 1°C.

The present study aimed to evaluate the response to salinity of Populus euphratica, which is more salt-resistant than other poplar cultivars, at the cellular level. To this purpose, callus was induced from shoot segments of P. euphratica on Murashige and Skoog (MS) medium supplemented with 0.5 mg l⁻¹ (2.2 μM) 6-benzyladenine (BA) and 0.5 mg l⁻¹ (2.7 μM) 1-naphthaleneacetic acid (NAA). Callus was transferred to MS medium supplemented with 0.25 mg l⁻¹ (1.1 μM) BA and 0.5 mg l⁻¹ NAA. The relative growth rate of callus reached a maximum in the presence of 50 mmol l⁻¹ NaCl and growth was inhibited with increasing NaCl concentrations. Examination of the changes of osmotic substances under salt stress showed that accumulation of proline, glycine betaine, and total soluble sugars increased with increasing salt concentrations. The results indicate that the response of the callus of P. euphratica to salt stress is similar to that of the whole plant.

The effect of abscisic acid (ABA) was evaluated during the maturation and germination of holm oak (Quercus ilex L.) somatic embryos. The addition of ABA to the culture medium significantly reduced unwanted recurrent embryogenesis in mature somatic embryos without affecting the germination of embryos subjected to stratification at 4°C. Stratification at 4°C for 2 mo. was the most efficient for stimulating somatic embryo germination of holm oak. The addition of 90 and 450 mM sucrose also improved germination, while higher sucrose concentrations were inhibitory.

In the present work, histological changes observed at the base of Eucalyptus globulus shoots in in vitro culture are described. Shoots were placed on solidified Murashige and Skoog medium containing half the original salt concentration, the complete vitamin composition, 9.8 μM indolebutyric acid (IBA), and 30 g l⁻¹ agar, and were incubated in the dark for the first 7 d, followed by a 16-h photoperiod. In vitro-generated roots could be originated either from old vascular tissue or from newly formed xylem. The influence of the preexistent tissues on the neoformation process appeared to be varied. The medulla did not intervene directly, although there were abundant cellular divisions in response to the induction medium. On the other hand, the interruptions observed in the vascular cylinder of the stem suggested an influence of the interfascicular parenchyma, and therefore the medulla could have participated in the differentiation process. However, the cortical parenchyma showed most of the changes that lead to the formation of adventitious roots of E. globulus growing in vitro. Histological analysis suggests that vascular rays can also be formed in direct contact with the central cylinder of the stem, although they mainly originate from the cortical parenchyma.

In vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l⁻¹ (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.

Adventitious root and shoot formation was obtained from cotyledon fragments of chestnut (Castanea sativa Mill.) and these processes followed two phases. In a first stage after detachment of the embryonic axis, the cotyledon fragments in culture formed a cotyledon petiole, which elongated for about 6 d. Thereafter, root primordia arose at the tip of the cotyledon petioles, followed by normal root development. In some cases, the cotyledon petioles showed adventitious shoot regeneration from a nodular structure previously formed at the end of the petioles. The presence or absence of growth regulators did not significantly influence root regeneration, whereas cytokinins stimulated shoot formation. The processes of root and shoot differentiation were studied also at the histological level. Observation with a light microscope showed that the developing root apical meristems were connected with a vascular bundle of the cotyledon petiole. Similarly, shoot bud meristem connections were observed with vascular tissue inside the nodular structure.

Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5× the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regenerated shoots. For long-term callus cultures, 2× KNO₃ or 4× Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.

The present study reports that a revised nutrient concentration in the basal medium improved shoot bud induction and subsequent plant regeneration in barley (Hordeum vulgare L. var. BL-2). Cultures were raised from immature embryos on MSB₅ medium supplemented with picloram. Concentrations of five nutrients were varied. The effect of these nutrients was investigated on (1) induction, (2) induction and subculture, and (3) induction, subculture and regeneration stages. The basal MSB₅ medium was not optimal for each phase of barley culture. Decreased ammonium nitrate, increased potassium dihydrogen phosphate, sodium molybdate, cobalt chloride, and addition of glycine enhanced shoot bud induction and plant regeneration. The different media that were optimal for immature embryo culture were: MSB₅ medium supplemented with 20.70 μM picloram, 10.30 mM NH₄NO₃, 6.25 mM KH₂PO₄, 2.06 μM Na₂MoO₄, 0.55 μM CoCl₂, and 26.64 μM glycine (for induction); MSB₅ medium supplemented with 12.47 μM picloram, 10.30 mM NH₄NO₃, and 0.55 μM CoCl₂ (for subculture); and MSB₅ medium supplemented with 0.2 μM picloram and 10.3 mM NH₄NO₃ (for regeneration). Primary cultures required 6 wk (without transfer) for morphogenic callus formation. Callus required 4 wk of subculture and another 4 wk on regeneration medium for optimal plant regeneration. The revised medium could also promote regeneration of the recalcitrant barley genotype RD-2552. Histological analysis showed that the major pathway of differentiation was through shoot bud formation.

Shi-hu (Dendrobium spp. or Dendrobii Herba) is one of the important traditional Chinese medicines. The commercially available crude drug in the traditional medicine market is composed mainly of three species: Dendrobium tosaense, D. nobile, and D. moniliforme. An efficient method of propagation has been developed via asymbiotic germination of seeds in vitro for the medicinally important D. tosaense. Seeds from capsules of D. tosaense collected 8–14 wk after artificial pollination germinated after being cultured on full-strength or half-strength Murashige and Skoog (MS) medium devoid of plant growth regulators and with 3% sucrose. Germination of seeds varied with the medium type and seed maturity. Germinated seedlings after transfer to MS medium with 1.5% sucrose and 8% banana homogenate or potato juice or coconut water and 20 wk of incubation developed into healthy plantlets. Well-developed plantlets were transplanted to moss or moss and tree fern or tree fern as substrates in plastic trays and transferred to a greenhouse for hardening. All plants survived, attained maturity, and developed normal flower and capsule after one and a half years. This protocol of successful plant regeneration by asymbiotic seed germination should permit rapid propagation and conservation of this medicinally important Dendrobium species.

Scientific photography is an important facet of plant tissue culture. The aim of photography in plant tissue culture should be to illustrate clearly the developmental stages occurring in vitro. However, the photographic results presented in publications are often poor, and morphogenetic responses are often not clearly documented. Plant tissue culture is a very visual science, and the valuable tool of photography is often not used properly. If the morphogenetic responses are not well documented, an important part of the research is missed, and the report ends up having limited scientific value. Simple methods for improving the results of photography in plant tissue culture are discussed, along with photographic equipment, photomacrography, stereophotomicrography, suitable backgrounds for photography, use of a digital scanner, and the construction of photographic plates.