Immature zygotic embryo has been the widely used explant source to develop embryogenic callus lines, cell suspensions and protoplasts for transformation of cereal crops including maize, wheat, rice, oat, barley, sorghum, and millet. However, the lack of competence of immature embryos in certain elite lines is still a barrier to routine production of transgenic cereal crops in certain commercial cultivars. In addition, a great deal of effort is required to produce immature embryos, manipulate cultures of immature embryos or their cell suspensions, and cryopreserve cultures for further use. In addition, undifferentiated cells may have reduced regenerability after a few months of in vitro culture. Alternative explants and regeneration systems for efficient transformation of cereal crops are needed to avoid or reduce the above limitations. During the past decade, scientists have successfully manipulated the shoot apical meristems from seedlings of maize, oat, sorghum, millet, wheat, and barley in an effort to develop a less genotype-dependent and efficient cereal regeneration system that can be maintained in vitro for long periods of time without the need for cryopreservation. Furthermore, apical meristem regeneration systems were used to stably transform maize, wheat, rice, oat, barley, sorghum, and millet.

Plant protoplasts provide an enabling technology to underpin aspects of development, physiology, and genetics. Reliable procedures are available to isolate and culture protoplasts from monocotyledons and dicotyledons. Several parameters influence the totipotency of protoplasts and their derived cells, particularly the source tissue, culture medium, and environmental factors. Novel approaches to maximize the efficiency of protoplast-to-plant systems include techniques already established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and artificial respiratory gas carriers, especially perfluorochemicals and hemoglobin. Somatic hybridization by protoplast fusion is undergoing a resurgence of interest, since it enables nuclear and cytoplasmic genomes to be combined at the interspecific and intergeneric levels without prior knowledge of gene location, or involvement of recombinant DNA technology. DNA uptake into protoplasts has applications in transient and stable transformation, including the generation of transplastomic plants of commercial importance in molecular pharming. Other applications of isolated protoplasts are in studies of membrane function, cell structure, and longer-term toxicological assessments. Despite the century that has elapsed since protoplasts were first isolated, they still make a significant contribution to many aspects of modern plant biotechnology.

Current methods for creating transgenic varieties are labor and time intensive, comprised of the generation of hundreds of plants with random DNA insertions, screening for the few individuals with appropriate transgene expression and simple integration structure, and followed by a lengthy breeding process to introgress the engineered trait into cultivated varieties. Various modifications of existing methods have been proposed to speed up the different steps involved in plant transformation, as well as a few add-on technologies that seek to address issues related to biosafety or intellectual property. The problem with an assortment of independently developed improvements is that they do not integrate seamlessly into a single transformation system. This paper presents an integrated strategy for plant transformation, where the introduced DNA will be inserted precisely into the genome, the transgenic locus will be introgressed rapidly into field varieties, the extraneous transgenic DNA will be removed, the transgenic plants will be molecularly tagged, and the transgenic locus may be excised from pollen and/or seed.

Although somatic hybridization techniques are being ignored by variety improvement programs for most commodities, their contribution to citrus variety improvement continues to expand and with increasing complexity. Citrus is one of the few commodities where somatic hybridization is reaching its predicted potential, as somatic hybrids are now possible from most desirable parental combinations. Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 130 at the CREC. The CREC hybrids include 34 from sexually compatible intergeneric combinations, 16 from sexually incompatible combinations, and 81 interspecific combinations. The objective of this report is to demonstrate the impact of somatic hybridization on citrus improvement programs, and to discuss its potential with other commodities. For citrus scion improvement, several applications are aimed at the development of improved seedless fresh fruit varieties, and these include symmetric somatic hybridization, haploid diploid fusion, targeted cybridization to transfer cytoplasmic male sterility (mtCMS) from Satsuma mandarin, and triploidy via interploid crosses using somatic hybrid allotetrapoid breeding parents. For rootstock improvement, symmetric somatic hybridization provides an opportunity to hybridize complementary rootstocks without breaking up successful gene combinations. Rootstock somatic hybridization is providing opportunities for improving disease and insect resistance, soil adaptation, and tree size control. Wide somatic hybridization provides an opportunity for gene transfer from related species, including some that are sexually incompatible. Extensive field research on citrus somatic hybrid rootstocks combined with emerging molecular analyses of citrus has allowed for the development of additional strategies for rootstock improvement. These include rootstock breeding and selection at the tetraploid level using somatic hybrid parents, and the resynthesis of important rootstocks at the tetraploid level via fusion of selected superior parents. Ongoing examples of each strategy will be provided, along with ideas for extending the technology to other commodities.

A procedure for protoplast isolation and plant regeneration of St. John's wort has been developed to utilize cell-to-cell variability for optimum production of valuable medicinal compounds. Calluses, induced from hypocotyl segments of St. John's wort seedlings, were used for protoplast isolation, induction of sustained cell division, and ultimately, plant regeneration. Callus-isolated protoplasts at a density of 2.0×10⁵ per ml were embedded in 0.6% Na-alginate blocks and cultured in a medium containing modified Murashige and Skoog (MS) salts, 2.5 μM 6-benzylaminopurine (BA), 5.0 μM α-naphthaleneacetic acid (NAA), and 0.5 mol l⁻¹ glucose. Protoplast-derived colonies formed compact calluses when transferred onto 0.35% gellan gum-solidified MS medium supplemented with 2.5 μM BA and 2.5 μM NAA. Shoot organogenesis from the protoplast-derived callus was induced on MS medium supplemented with 5 μM thidiazuron. Complete plantlets were obtained from the regenerated shoots on MS basal medium. A greater than 3-fold variation of antioxidant activity was observed among the protoplast-derived plantlets and chemically distinct germplasm lines were selected on the basis of phytochemical profiles. The protoplast to plant regeneration protocol developed in this study provides the foundation for development of novel genotypes with potential expansion of the genetic diversity through somatic hybridization, and organelle transplantation.

Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V.×Labruscana L.H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay,’ ‘Merlot,’ and ‘Pinot Noir’ occurred on medium C \[Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol, 0.3% (w/v) Phytagel, 2.5 μM 2,4-dichlorophenoxyacetic acid, 2.5 μM β-naphthoxyacetic acid, 5.0 μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine\]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay,’ ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.

Germination of tamarind seeds in medium containing thidiazuron (TDZ) resulted in induction of nodular protrusions in and around the cotyledonary node meristem. The structures developed radially in well-defined circles and subsequently spread towards the cotyledonary bridge and also in the proximal part of the hypocotyl. The structures developed into shoots on transfer to medium devoid of growth regulators. Histological studies revealed that the protrusions initiated from the nodal meristem and extended to the non-meristematic region between the two meristems and also in the proximal part of the hypocotyl in seedlings germinated in 9.08 μM TDZ. Newly formed cell layers and less-differentiated meristematic protrusions were also seen. With the increase in the distance from the meristem, the buds were less differentiated; in the proximal part of the hypocotyl only the multiple layers of meristematic cells were noted. With extension of the period of incubation, the TDZ-induced meristematic activity extended laterally in circles towards the neighboring region. The radial spread of the meristematic activity from the center of the nodal meristem was also evident at 18.16 μM TDZ. From the pattern of the morphogenic development and the histological studies it may be hypothesized that in tamarind, TDZ influences the existing meristems specifically. Subsequently de novo organogenesis is triggered in the neighboring cells.

This is the first report of simultaneous organogenesis and somatic embryogenesis in Arnebia euchroma, a highly valued, critically endangered medicinal plant of the Himalaya. Root-derived callus showed only rhizogenesis, whereas leaf-derived callus showed simultaneous organogenesis and somatic embryogenesis. Organogenesis was optimal (12.2 shoots per culture) in 1 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine and induction of somatic embryogenesis (16.3 embryos per culture) occurred in 2.5 μM indole-3-butyric acid combined with 2.5 μM 6-benzyladenine. Shoots rooted (100%) best in half-strength Murashige and Skoog (MS) medium supplemented with 2.0 μM indole-3-butyric acid. Early cotyledonary-stage embryos encapsulated with 3% sodium alginate and calcium nitrate (100 mM for 25 min) showed 60.6% germination in MS medium. Rooted shoots transferred to a mixture of sterile soil, sand, and peat (1:1:1 by volume) showed 72% survival ex vitro. Application of these protocols would be helpful in reducing pressure in natural populations, in genetic transformation studies, and in long-term storage of elite genotypes through synthetic seed production.

An in vitro protocol has been developed for callus induction, somatic embryogenesis, and plant regeneration from stigma–style culture of grapevine. Four different grapevine cultivars (Vitis vinifera L.; cvs. ‘Bombino Nero,’ ‘Greco di Tufo,’ ‘Merlot,’ and ‘Sangiovese’) were tested. Explants were cultured on Nitsch and Nitsch medium (NN) supplemented with various combinations of 6-benzylaminopurine (BA; 4.5 and 9.0 μM) and β-naphthoxyacetic acid (NOA; 5.0 and 9.9 μM). Sucrose (88 mM) was used as the carbon source. Somatic embryogenesis was induced within 3–7 mo. after culture initiation. Even though explants of different origin (unfertilized ovules and anthers) regenerated somatic embryos, the higher embryogenic potential was observed in stigma and style explants, with the exception of ‘Merlot,’ which regenerated somatic embryos only from unfertilized ovules. The percentages of stigma–style explants producing somatic embryos was 7% in ‘Bombino Nero’ (cultured on NN medium supplemented 9.0 μM BA and 9.9 μM NOA), 14% in ‘Greco di Tufo’ (4.5 μM BA and 9.9 μM NOA), and 8% in ‘Sangiovese’ (9.0 μM BA and 9.9 μM NOA). The presence of growth regulators (BA and NOA) in the medium was essential for induction of somatic embryogenesis. Plants were regenerated on hormone-free NN medium containing 88 mM sucrose.

Regeneration of plants via somatic embryogenesis was achieved from zygotic embryo explants isolated from mature seeds of Schisandra chinensis. Merkle and Sommer's medium, fortified with 2,4-dichlorophenoxyacetic acid (2,4-D; 9.04 μM) and zeatin (0.09 μM), was effective for induction of embryogenic callus. The development of a proembryogenic mass and somatic embryos occurred on Murashige and Skoog medium (MS) free of plant growth regulators. The embryogenic callus induced on Merkle and Sommer's medium supplemented with 2,4-D (9.04 μM) and zeatin (0.09 μM) showed development of the maximum number of somatic embryos when transferred to MS medium free of plant growth regulators. The maximum maturation and germination of cotyledonary somatic embryos (46.3%) occurred on MS medium supplemented with 2,4-D (0.45 μM) and N⁶-benzyladenine (1.11 μM). The somatic embryo-derived plants were successfully hardened, with a survival rate of approximately 67%, and established in the field.

Guar gum, a galactomannan derived from the endosperms of Cyamopsis tetragonoloba, has been successfully used as a sole gelling agent for plant tissue culture media. Its suitability as a gelling agent was demonstrated by using guar gum-gelled media for in vitro seed germination of Linum usitatissimum and Brassica juncea, in vitro axillary shoot proliferation in nodal explants of Crataeva nurvala, rooting of regenerated shoots of the same, in vitro androgenesis in anther cultures of Nicotiana tabacum, and somatic embryogenesis in callus cultures of Calliandra tweedii. The media used for these were gelled with either guar gum (2, 3, or 4%) or agar (0.9%). Guar gum-gelled media, like agar media, supported all these morphogenic responses. Rather, axillary shoot proliferation, rhizogenic and embryogenic responses were better on guar gum-gelled media than on agar media.

Camptothecin, produced by Camptotheca acuminata, is a pharmaceutically important compound. Transgenic technology has potential uses for the enhancement of camptothecin production; however, an efficient plant regeneration protocol for C. acuminata is not currently available. Factors that affected successful seedling germination were evaluated. The regeneration potential of various parts of seedlings was tested. Camptothecin production in regenerated plants was compared to its production in calluses and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's B5 medium 8.9 μM benzyladenine. Among the calluses induced from various parts of seedlings, leaf petiole calluses, leaf disc calluses, and cotyledon calluses regenerated shoots, but internode calluses did not. Furthermore, leaf petiole calluses and leaf disc calluses regenerated normal shoots, while cotyledon calluses regenerated hyperhydric shoots. Moreover, leaf petiole calluses had a higher shoot regeneration rate, 50% versus 9%, and a higher shoot number, 6.2 ± 0.5 versus 2.0 ± 0.3, than did leaf disc calluses on the best shoot induction medium. It took 4–6 wk to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calluses and similar to that in the original seedlings. In conclusion, leaf petioles provide efficient plant regeneration of C. acuminata.

The influence of donor plant growth environment, microspore development stage, culture media and incubation conditions on microspore embryogenesis was studied in three Indian B. juncea varieties. The donor plants were grown under varying environments: field conditions, controlled conditions, or a combination of the two. The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same does not hold true for the field-grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive with genotypic variability ranging from 10 to 35 embryos per Petri dish, irrespective of the other factors. NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis. The fortification of this medium with activated charcoal, polyvinylpyrrolidone, colchicine, or growth regulators (6-benzylaminopurine and 1-naphthaleneacetic acid) was observed to be antagonistic for microspore embryogenesis, while silver nitrate (10 μM) had a significant synergistic effect. A post-culture high-temperature incubation of microspores at 32.5 ± 1°C for 10–15 d was found most suitable for high-frequency production of microspore embryos. The highest frequency of microspore embryogenesis (78 embryos per Petri dish) was observed from the late uninucleate microspores (contained in bud sizes 3.1–3.5 mm irrespective of genotype) cultured on NLN medium containing 13% sucrose and silver nitrate (10 μM), and incubated at 32.5°C for 10–15 d.

Tissue culture and plant regeneration protocols for the salt marsh plants Juncus roemerianus Scheele and Juncus gerardi Loisel. were developed. J. roemerianus callus was induced from mature seeds cultured on Murashige and Skoog (MS) medium supplemented with 2.22 μM 6-benzylaminopurine (BA), 5.37 μM α-naphthaleneacetic acid (NAA), 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and 50 ml l⁻¹ coconut water (callus induction medium). The callus was subcultured on MS medium containing 2.22 μM BA, 5.37 μM NAA, and 9.05 μM 2,4-D for callus maintenance. Shoot regeneration occurred 2 wk after transferring the callus onto shoot regeneration medium, which consisted of MS medium containing BA or thidiazuron. A high frequency of shoot regeneration was obtained when the medium contained 13.3 μM BA. Regenerated shoots were transferred to MS medium supplemented with 10.7 μM NAA for root production. Rooting did not occur in the shoots regenerated on the thidiazuron-containing media. The callus induction medium for J. roemerianus was also effective in inducing callus of J. gerardi from young inflorescences. The same medium was also used for callus maintenance. Shoot regeneration occurred 10 d after transferring the callus onto MS medium supplemented with 0.44 μM BA and 0.57 μM indole-3-acetic acid. Root regeneration occurred after transferring the shoots onto MS medium plus 0.44 μM BA and 14.8 μM indole-3-butyric acid. The regenerated plants of both J. roemerianus and J. gerardi grew vigorously in potting soil in the greenhouse. J. roemerianus regenerants also grew well in a saltwater-irrigated field plot. Tissue culture-produced plants of J. roemerianus and J. gerardi can be used for planting in created or restored wetlands.

Plant tissue culture media, sterilized through commercial filter membrane units, inhibited plant cell growth and development. Embryogenesis was suppressed in microspore cultures of Brassica napus and anther cultures of Triticum aestivum, and in vitro pollen development was suppressed in B. napus. Inhibition of growth and development occurred on media that had been filter-sterilized in low volumes through non-washed filter membrane units of all brands tested, including Nalgene, Corning, Millipore, and Sartorius membrane products. Similar results were obtained with Costar Transwells and Millipore inserts (Millicell-CM, -HA, and -PCF). These deleterious effects were eliminated by washing the membranes with water followed by a small volume of the medium. Media sterilized by filtration through washed filters produced significant improvements of growth and development of embryos and pollen.

Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br. (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might offer an alternative method for production of these important pharmaceuticals, which would reduce the collection pressure on this rare plant.

Shoot regeneration in hairy root cultures of Solanum khasianum Clarke influences root growth, solasodine production, and permeabilization of solasodine into the medium. These parameters are dependent on exogenously supplied auxin and cytokinin; the effect being both concentration- and clone-dependent. Hairy root cultures with no shoot regeneration showed high permeabilization of solasodine into the medium by the sixth week of incubation, suggesting the medium acts as a sink for the solasodine synthesized by the roots. Solasodine in the culture medium was toxic to the transformed roots and caused browning of root tips. In a separate set of experiments, the hairy root cultures showed regeneration of approximately 50–70 mm long shoots after treatment with indole-3-acetic acid and kinetin. These hairy root cultures had increased levels of solasodine production, compared to cultures without shoot regeneration. The plantlets formed in the hairy root cultures accumulated some of the solasodine, thereby reducing its permeabilization into the medium. Transport of solasodine from root to shoot reduced the toxic effect of solasodine in the root zone and extended the exponential growth phase by 8–10 d.

In vitro adventitious shoot bud regeneration systems are considered most suitable for Agrobacterium- and biolistics-mediated genetic transformation to obtain transgenic plants. In the present investigation, multiple adventitious shoot buds could be induced directly from Feronia limonia hypocotyl explants inoculated on Murashige and Skoog (MS) medium containing different growth regulators. During the initial phase, the hypocotyl segments nearer to the cotyledons responded quickly compared to those closer to the root. The response, however, was comparable in both the segments in subsequent subculture. Of the various cytokinins, 2.22 μM 6-benzylaminopurine (BA) proved to be more effective compared to kinetin (Kn). The two-way interaction of BA and Kn significantly influenced shoot regeneration and contributed the most among the interactions studied. The best response, however, was obtained when 2.22 μM BA and 2.32 μM Kn were combined. Although the effect of auxins like α-naphthaleneacetic acid (NAA) combined with cytokinins evoked a significant response in terms of number of shoot buds, this response did not supersede the effect of combined cytokinins. None of the polyamines tested induced shoot buds on hypocotyl segments. Adventitious shoots were multiplied on MS medium containing 2.22 μM BA, 6.96 μM Kn, and 0.05 μM NAA. More than 60% of the shoots produced roots when cultured on medium containing one-quarter strength MS salts, 1.0% sucrose, 0.6% agar, and 7.36 μM indole-3-butyric acid. The adventitious origin of shoot buds showing continuous vascular connections was confirmed through histological investigations.

Kalopanax pictus (Thunb.) Nakai is a tall tree, and its wood has been used in making furniture, while its stem bark is used for medicinal purposes. Here, we report on the micropropagation of Kalopanax pictus via somatic embryogenesis. Embryogenic callus was induced from immature zygotic embryos. The frequency of embryogenic callus induction is influenced by days of seed harvest. Callus formation was primarily observed along the radicle tips of zygotic embryos incubated on Murashige and Skoog (MS) medium with 4.4 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryogenesis was observed following transfer of embryogenic callus to MS medium lacking 2,4-D. Somatic embryos at the cotyledonary stage were obtained after 6 wk following culture. Frequency of conversion of somatic embryos into plantlets was low (35%) on a hormone-free MS basal medium, but it increased to 61% when the medium was supplemented with 0.05% charcoal. Gibberellic acid (GA₃) treatment markedly enhanced the germination frequency of embryos up to 83%. All plantlets obtained showed 98% survival on moist peat soil (TKS2) artificial soil matrix. About 30 000 Kalopanax pictus plants were propagated via somatic embryogenesis and grown to 3-yr-old plants. These results indicate that production of woody medicinal Kalopanax pictus plantlets through somatic embryogenesis can be practically applicable for propagation.

Leaf structure along the successive stages of Early French artichoke Cynara scolymus L. micropropagation was characterized using light and transmission electron microscopy. The mesophyll presents disorganized spongy and palisade parenchyma with large intercellular spaces and a few small chloroplasts in the leaves of plants cultured in vitro. In addition, both epidermal surfaces of such leaves invariably show a cell wall of the same thickness with a very thin cuticle and open stomata. In the root differentiation stage in vitro, structural changes take place in the leaves that are favorable for survival in the acclimatization stage: conspicuous cuticle, greater cell wall thickness, functional stomata, better mesophyll organization, developed vascular bundles, and the presence of sclerenchymatous tissue are observed. These features found in later in vitro stages are maintained in the following ex vitro stages, some becoming more evident. Our results demonstrate that the structural changes required to ensure appropriate acclimatization of micropropagated artichoke plants begin at the root differentiation stage, which can reduce in vivo acclimatization time and achieve greater survival of transferred plants.

Somatic embryo (bipolar) or shoot (monopolar) morphogenesis in mesophyll cells of Euphorbia nivulia Buch.-Ham in vitro was dependent on the type of auxin supplementing Murashige and Skoog (MS) medium containing benzyladenine. Direct in vitro morphogenesis, i.e., organogenesis, and somatic embryogenesis were significantly influenced by seasonal growth of the donor plant, explant position (proximal, mid, and distal), and light. Explants collected in March/April were superior to July/August material. Proximal explants underwent morphogenesis more readily than mid- and tip-derived explants. Incubation in the light favored morphogenesis while darkness was inhibitory. Kinetin (Kn) was also inhibitory to morphogenesis. MS medium enriched with different levels of N⁶-benzyladenine (BA) alone, or in combination with α-naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA), induced adventitious shoots directly. Explants collected in March/April cultured on medium with 13.3 μM BA and 2.69 μM NAA developed the highest number of shoots, a mean of 15.2 shoots per proximal explant. Developed shoots rooted the best on half-strength MS medium with 2.46 μM indole-3-butyric acid, which developed a mean of 5.2 roots per shoot. Rooted healthy shoots could be transplanted to small pots, with an 80% survival rate. Addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to BA-supplemented medium was obligatory to develop somatic embryos. MS medium containing 2.26 μM 2,4-D and 4.44 μM BA induced a mean of 44.8 somatic embryos per proximal explant. The embryos passed through distinct stages of embryogenesis, namely globular, heart, torpedo, and early cotyledonary. The embryos (88%) underwent maturation on half-strength MS medium with 2.89 μM gibberellic acid (GA₃), and its subsequent transfer on half-strength MS basal medium in light conditions facilitated 80% conversion of embryos to plantlets. Direct shoots or embryos were originated from the mesophyll cells. Somatic embryo development was concurrent with the independent origin of vasculature in the bulbous basal portion. The survival rate of embryo-derived plants was 90%.

A protocol for micropropagation of plants via axillary bud proliferation from nodal explants of Terminalia bellirica Roxb. seedlings has been established. Explants were cultured on Murashige and Skoog medium with different concentrations of 6-benzyladenine (BA; 4.4, 8.9, 13.3, 17.8, or 22.2 μM) or kinetin (Kn; 4.6, 9.3, 14.0, 18.6, or 23.2 μM). Within the range evaluated, the medium containing 13.3 μM BA showed the highest shoot length (1.9 ± 0.2 cm) in the primary culture. When separated and transferred to fresh subculture medium with lower levels of BA (2.2, 4.4, 6.6, or 8.9 μM) or Kn (2.3, 4.6, 6.9, or 9.3 μM), the nodal segments from individual regenerants (obtained initially from seedling nodes) showed efficient shoot induction at 4.4 μM BA. Rooting of the shoots was achieved under in vitro conditions on two media tested, i.e., modified Gamborg's (B₅) medium or Woody Plant Medium, both supplemented with 4.9 μM indole-3-butyric acid. Regenerated plants were established in the greenhouse.

In vitro collecting is the process of initiating tissue cultures in the field. In order for in vitro collecting to be broadly available as a technique for collecting plant germplasm, the levels of contamination in such cultures must be controlled. Two techniques for in vitro collecting were compared: leaf punch and needle collecting. The effectiveness of these methods for collecting leaf and stem tissues from plants at tropical and temperate sites was compared. Stem tissue collected by the needle collecting method gave cultures with an average contamination percentage of 31% and 16%, from the tropical and temperate sites, respectively, while with the leaf punch method, average contamination percentages were 90% and 69%. The effectiveness of antimicrobial agents in reducing contamination in leaf punch cultures was evaluated. Addition of the fungicide benlate and the antibiotics, cefotaxime and vancomycin, to the leaf punch collections reduced contamination to an average of 30% in the tropical collections and 35% in the temperate collections. Over 90% of both tropical and temperate species collected in multiple samples of 10 or more had at least one clean sample using this medium. The use of either the leaf punch method in combination with a fungicide and antibiotics or the needle collecting technique yielded a high percentage of clean tissues for study and growth.

An efficient and reproducible procedure for the large-scale propagation of Pseudoxytenanthera stocksii is described. High-frequency multiple shoot induction was achieved from nodal shoot segments collected from superior/elite genotypes on Murashige and Skoog (MS) liquid medium supplemented with 1-naphthaleneacetic acid (NAA; 2.68 μM) and 6-benzylaminopurine (BA; 4.40 μM) at 28 ± 1°C and 60 μmol m⁻² s⁻¹ light intensity under 12 h photoperiod. In vitro-differentiated shoots were multiplied on MS liquid medium fortified with NAA (2.68 μM), BA (2.21 μM) and additives: ascorbic acid (283.93 μM), citric acid (118.10 μM), cysteine (104.04 μM), and glutamine (342.24 μM). Subculturing was carried out every 2 wk on fresh shoot multiplication medium. About 125–150 shoots per culture flask were harvested within 45–50 d. In vitro-differentiated shoot clumps (three or four shoots) were successfully rooted on half-strength MS basal liquid medium with indole-3-butyric acid (4.90 μM), BA (0.44 μM), and additives. This is the first report where in vitro-and in vivo- (through tillers) raised clonal plants were acclimatized and established in the field, where they exhibited normal growth.

Yew cell suspension culture is used as an alternative source of paclitaxel, an anti-cancer drug. To optimize the initiation protocol, highly dormant yew seeds were germinated in vitro and the seedlings used to establish callus culture. The best sources of explant for callus initiation and growth were seedling stems and roots, and the most successful medium was modified B5 medium containing 2,4-dichlorophenoxyacetic acid and kinetin. Calluses were friable and suitable for establishing cell suspension cultures, which were maintained for over 3 yr. Flow cytometric analysis of nuclear DNA content revealed that 2-yr-old cell suspension cultures consisted predominantly of putative euploid and aneuploid cells coexisting as sub-populations. Additional measurements performed 3 and 7 mo. later revealed further genomic instability, with a tendency towards a higher proportion of cells with elevated nuclear DNA content. In a selected cell line, which showed significant taxane production, the addition of 100 μM jasmonic acid strongly enhanced total taxane production and slightly inhibited growth while no effect on nuclear DNA content was noted.