The capacity of cultured plant tissues and cells to undergo morphogenesis, resulting in the formation of discrete organs or whole plants, has provided opportunities for numerous applications of in vitro plant biology in studies of basic botany, biochemistry, propagation, breeding, and development of transgenic crops. While the fundamental techniques to achieve in vitro plant morphogenesis have been well established for a number of years, innovations in particular aspects of the technology continue to be made. Tremendous progress has been made in recent years regarding the genetic bases underlying both in vitro and in situ plant morphogenesis, stimulated by progress in functional genomics research. Advances in the identification of specific genes that are involved in plant morphogenesis in vitro, as well as some selected technical innovations, will be discussed.

The cucurbit family includes a number of valuable crop species (melon, cucumber, squash/pumpkin, watermelon). Much of this review is concerned with transgenic resistance to viruses, shown to be the major application of biotechnology in the cucurbit family. Progress made with the production of transgenic cucurbit crops is discussed. Published data on field tests of transgenic cucurbits are reviewed, showing that much progress has been made with multiple virus-resistant cucurbit crops which can be productive without chemical control of insect virus vectors. Modes of virus resistance in transgenic cucurbits are discussed, as is the bio-safety of such crops. For the first time a detailed analysis has been made of world-wide and US field test applications for cucurbit crops. World-wide, most field test applications were for melon (54%), followed by squash (32%). World-wide most field test applications were for virus resistance (84%), and most applications (77%) were in the USA. Two transgenic multiple virus-resistant squash crops have been deregulated (released for sale). Additionally, the analysis shows that there are transgenic multiple virus-resistant crops in all major cucurbit species already available, for which several different companies have applied for field tests. This would imply that such crops are ready to be marketed should conditions permit, which would have an impact world-wide in reduction of ecological damage due to chemical control of the insect viral vectors.

To achieve reliable stable transformation of sweet potato, we first developed efficient shoot regeneration for stem explants, leaf disks, and petioles of sweet potato (Ipomoea batatas (L.) Lam.) cultivar Beniazuma. The shoot regeneration protocol enabled reproducible stable transformation mediated by Agrobacterium tumefaciens strain EHA105. The binary vector pIG121Hm contains the npt II (pnos) gene for kanamycin (Km) resistance, the hpt (p35S) gene for hygromycin (Hyg) resistance, and the gusA (p35S) reporter gene for β-glucuronidase (GUS). After 3 d co-cultivation, selection of calluses from the three explant types began first with culture on 50 mg l⁻¹ of Km for 6 wk and then transfer to 30 mg l⁻¹ of Hyg for 6–16 wk in Linsmaier and Skoog (1965) medium (LS) also containing 6.49 μM 4-fluorophenoxyacetic acid and 250 mg l⁻¹ cefotaxime in the dark. The selected friable calluses regenerated shoots in 4 wk on LS containing 15.13 μM abscisic acid and 2.89 μM gibberellic acid under a 16 h photoperiod of 30 μmol m⁻² s⁻¹. The two-step selection method led to successful recovery of transgenic shoots from stem explants at 30.8%, leaf discs 11.2%, and petioles 10.7% stable transformation efficiencies. PCR analyses of 122 GUS-positive lines revealed the expected fragment for hpt. Southern hybridization of genomic DNA from 18 independent transgenic lines detected the presence of the gusA gene. The number of integrated T-DNA copies varied from one to four.

An elite aspen hybrid (Populus×canescens×P. grandidentata) was transformed with Agrobacterium tumefaciens strain EHA105 that harbored a binary vector (pBI121) carrying the nptII gene under the nos promoter and tandem rolB–uidA (GUS) genes with the CaMV 35S or heat shock promoter. Among 32 independent kanamycin-resistant plants, 25 plants were confirmed by polymerase chain reaction and Southern blot analyses to contain all three genes, whereas five plants contained only nptII or/and uidA genes and two plants had both the rolB and nptII or uidA genes. Integration of the rolB gene significantly increased rooting ability of hardwood cuttings. Heat shock-rolB-transformed plants rooted at significantly higher percentage than the CaMV 35S-rolB-transformed plants. Heat shock treatment further enhanced rooting of heat shock-rolB-transformed plants. Exposure to exogenous auxin did not significantly increase the rooting percentage of transgenic hardwood cuttings, but increased the number of roots induced. This research shows great potential to improve rooting of hardwood cuttings of difficult-to-root woody plants which are commercially important to the horticultural and forestry industry. The transgenic plants with gain-of-function in hardwood-cutting rooting can facilitate research in the understanding of adventitious rooting from hardwood cuttings of recalcitrant woody plants.

A new protocol has been developed for the highly efficient somatic embryogenesis and plant regeneration of 10 recalcitrant Chinese cotton cultivars. Calluses and embryogenic calluses were induced on MSB1 medium containing the optimal combination of indolebutyric acid (IBA; 2.46 μM) and kinetin (KT; 2.32 μM). Up to 86.7% of embryogenic calluses differentiated into globular somatic embryos 2 mo. after culture on MSB2 medium containing double KNO₃ and free of growth regulators. Up to 38.3% of the somatic embryos were converted into complete plants in 8 wk on MSB3 medium with l-asparagine (Asn)/l-glutamine (Gln) (7.6/13.6 mM). The plants were successfully transferred to soil and grew to maturity. With the protocol described here, we have obtained hundreds of regenerating plantlets from 10 recalcitrant cultivars, which is important for the application of tissue culture to cotton breeding and biotechnology.

High-frequency embryogenesis systems were established for hybrid yellow-poplar (Liriodendron tulipifera×L. chinense) and hybrid sweetgum (Liquidambar styraciflua×L. formosana) by modifying a medium originally developed for embryogenic yellow-poplar cultures. Embryogenic cultures of both hybrids, consisting of proembryogenic masses (PEMs), were initiated from immature hybrid seeds on an induction-maintenance medium (IMM) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and casein hydrolyzate (CH). For hybrid yellow-poplar, as many as 2100 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM lacking CH, at a pH that varied with genotype (3.5 or 5.6), followed by size fractionation and plating on semisolid embryo development medium (DM; IMM lacking 2,4-D and BA) without CH, but supplemented with 4.0 mg l⁻¹ (15 μM) abscisic acid. For hybrid sweetgum, up to 1650 germinable somatic embryos per 4000 cells or cell clumps were produced when PEMs were grown in liquid IMM without CH, but with 550 mg l⁻¹ l-glutamine, 510 mg l⁻¹ asparagine, and 170 mg l⁻¹ arginine at pH 5.6. Somatic embryos developed from cell clumps on DM without any plant growth regulators or other supplements. Hundreds of somatic embryos of both hybrids were germinated on DM without CH, transferred to potting mix, and hardened off in a humidifying chamber for transfer to the greenhouse.

The effect of different sealing materials \[i.e., polyvinyl chloride (PVC) transparent film, and Parafilm (PARA)\] for Petri dishes was investigated on shoot regeneration from quince (Cydonia oblonga L.) ‘BA 29’ leaf explants. Leaves were excised from proliferating shoot cultures, transversally scored, and placed with the abaxial side down in 60-mm Petri dishes containing 10 ml of Murashige and Skoog modified medium, with 5.4 μM α-naphthaleneacetic acid, 4.5 μM thidiazuron, 200 mg l⁻¹ cefotaxime, and 0.25% (w/v) Phytagel (IM medium) for shoot bud induction, and cultured in darkness at 22 ± 2 °C for 28 d. Then the explants were transferred to standard conditions (16-h photoperiod at 30 μmol m⁻² s⁻¹ photosynthetically active radiation) on a medium similar to IM, except for lack of NAA, and with 0.65% (w/v) agar instead of Phytagel, for an additional 15–28 d. The sealing combinations PARA–PARA, PARA–PVC, PVC–PARA, and PVC–PVC (in the induction–expression phases) were compared during regeneration and for their carry-over effect on shoot development after transfer of explants to an elongation medium (0.9 μM 6-benzyladenine). Carbon dioxide accumulated at 27.2 mmol mol⁻¹ at the end of induction, and gradually decreased from 35.4 mmol mol⁻¹ on day 9 to 22.5 mmol mol⁻¹ on day 28 of the expression phase in PARA-sealed Petri dishes, being always much higher than after sealing with PVC (1–2 mmol mol⁻¹). Ethylene concentration was 0.1 and 0.04 μmol mol⁻¹ in the first part of the induction and expression phase, respectively, in PARA-sealed Petri dishes, and slightly decreased with duration of exposure to light during expression; while it was absent in most PVC-sealed dishes. The PARA–PARA and PVC–PVC (induction–expression) combinations gave, respectively, the worst and best results of regeneration and successive shoot development.

Cryopreservation of African violet via encapsulation–dehydration, vitrification, and encapsulation–vitrification of shoot tips was evaluated. Encapsulation–dehydration, pretreatment of shoot tips with 0.3 M sucrose for 2 d followed by air dehydration for 2 and 4 h resulted in complete survival and 75% regrowth, respectively. Dehydration of encapsulated shoot tips with silica gel for 1 h resulted in 80% survival but only 30% regrowth. Higher viability of shoot tips was obtained when using a step-wise dehydration of the material rather than direct exposure to 100% plant vitrification solution (PVS2). Complete survival and 90% regrowth were achieved with a four-step dehydration with PVS2 at 25°C for 20 min prior to freezing. The use of 2 M glycerol plus 0.4 M sucrose or 10% dimethyl sulfoxide (DMSO) plus 0.5 M sucrose as a cryoprotectant resulted in 55% survival of shoots. The greatest survival (80–100%) and regrowth (80%) was obtained when shoot tips were cryoprotected with 10% DMSO plus 0.5 M sucrose or 5% DMSO plus 0.75 M sucrose followed by dehydration with 100% PVS2. Shoot tips cryoprotected with 2 M glycerol plus 0.4 M sucrose for 20 min exhibited complete survival (100%) and the highest regrowth (55%). In encapsulation–vitrification, dehydration of encapsulated and cryoprotected shoot tips with 100% PVS2 at 25°C for 5 min resulted in 85% survival and 80% regrowth.

The effect of ventilation during the multiplication stage on the development of propagules from different clones of jojoba \[Simmondsia chinensis (Link) Schneider\] was investigated. Variation in the response to ventilation was due to genotype, the extent of ventilation, and to the period of exposure (transfer number). With intermediate ventilation treatments, propagules elongated to a greater extent and produced more dry biomass than propagules grown without ventilation. In the highest ventilation treatment, however, growth parameters were negatively affected. More importantly, propagules grown with moderate ventilation produced more plant material suitable for further multiplication and for the elongation stage than those grown in sealed tubes – the vessels used in our original micropropagation system. In five of the seven clones studied, growth and multiplication rate were decreased by the highest ventilation treatment. Propagules from the second and third multiplication transfers into ventilated vessels became more sensitive to high ventilation. Ambient water loss was slower in propagules produced under ventilation, probably due to smaller stomatal apertures. As a result of improved growth and decreased hyperhydricity by ventilation, the micropropagation protocol should be modified to include Magenta boxes equipped with vented lids as the preferred growing vessels.

An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indole-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted micro-shoots were transferred to a coco-peat, perlite, and vermiculite (1:1:1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation–dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1 h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.

Studies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.

A protocol for large-scale propagation of Phragmites communis Trin. by adventitious bud formation and plant regeneration was established. Adventitious buds were induced through either the indirect pathway or the direct pathway from stem explants of Phragmites communis. In the indirect pathway, it was essential to decrease the level of 2,4-dichlorophenoxyacetic acid from 9.1 to 0.5 μM to induce adventitious buds and achieve plant regeneration. In the direct pathway, the effects of different benzylaminopurine (BA) concentrations in the medium, and different positions of the explants, on adventitious bud formation were determined. Murashige and Skoog (MS) medium supplemented with 5.4 μM α-naphthaleneacetic acid (NAA) and 53.4 μM BA, and the bottom part of stem explants were most responsive for the differentiation of adventitious shoot buds. The highest differentiation frequency was 20–30 adventitious shoot buds per stem node tissue. Elongation and proliferation of adventitious buds were achieved on MS medium supplemented with 13.3 μM BA and 5.4 μM NAA. Shoots were rooted in liquid half-strength MS medium with 5.4 μM NAA 4.9 μM indole-3-butyric acid. Rooted plants survived (87.5%) and grew well after transfer into soil for 4 wk. More than 20 000 regenerated plants of a salt-tolerant variant line of Phragmites communis have been produced. This protocol is useful for clonal micropropagation and possibly for Agrobacterium- mediated gene transfer in P. communis.