Morphological and agronomic description of selectedCuphea germplasm

Forty Cupheaspecies, recently collected in mid- and southern Mexico, are described in terms of their main morphological and agronomic characteristics. In some species, dormancy, slow germination, sticky hairs, and perennial character, as well as poor flowering and low seed yield limit their agronomic potential. However, some species of the section Heterodon show good crop potential. Among these, C. wrightii, C. glossostoma,and C. laminuligeraseem to be the best lauric acid-synthesizing species. Of the capric acid species, C. paucipetala, C. lanceolata,and C. procumbenshave the best growing potential in humid climates, whereas C. leptopodaand C. inflataare well adapted to arid growing conditions. All Cupheaspecies show seed shattering caused by flower zygomorphy. This zygomorphy not only causes different petal shape and a bilateral symmetry of the calyx tube, but also dispersal of the seeds. It represents the main taxonomic trait for characterizing the genus. Flowering and seed setting continue over a period of several weeks due to indeterminate growth. Multiple harvests with vacuum picking systems that do not damage the plants maximize recovered yields.     

An efficient in vitro plantlet regeneration of <Emphasis Type="Italic">Cryptocoryne wendtii</Emphasis> and <Emphasis Type="Italic">Cryptocoryne becketti</Emphasis> through shoot tip culture

An efficient micropropagation protocol was established for Cryptocoryne wendtii and Cryptocoryne becketti using shoot tips explants. Multiple shoots were induced from shoot tip explants of both species cultured on agar-gelled as well as liquid MS medium supplemented with 0.5 mg/L BA and 0.2 mg/L IBA (proliferation medium). The multiple shoots of both the species formed on agar-gelled as well as liquid medium were vigorously growing with well-developed roots and leaves after 4 weeks of culture. Highest number of multiple shoots was obtained from shoot tip explants of both the species cultured in liquid proliferation medium after 4 weeks of culture. The shoot tip explants of C. wendtii and C. becketti, that were cultured in liquid proliferation medium (2 weeks) followed by culturing on agar-gelled proliferation medium (2 weeks) also produced the multiple shoots. Shoot tips cultured on agar-gelled medium produced the least number of multiple shoots after 4 weeks of culture. Histological study did not show any abnormalities in the leaves of in vitro plantlets of both the species, cultured in agar-gelled and liquid proliferation medium. The leaves of the in vitro plantlets formed normal mesophyll cells and vascular bundles. More than 95% of the acclimatized plantlets grew vigorously without any morphological abnormalities.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Survival and Expansion of Mechanically Transplanted Seagrass Sods

Although planting seagrass is not technically complex, the ability to plant large areas is limited by the time-consuming nature of manual methods. Additionally, manual methods use small, spatially isolated planting units (PUs; shoot bundles or plugs/cores) that are often highly susceptible to disturbance. The likelihood for harvesting intact apical meristems may be higher with large sods compared to smaller units, thus increasing survival and expansion rates. Here, we examined the survival and expansion of large units (1.5 × 1.2 m) of seagrass transplanted using a mechanized planting boat (Giga Unit Transplant System; GUTS). Twenty-seven units of seagrass (18 Halodule wrightii and 9 Thalassia testudinum ) were transplanted and monitored for survival, shoot density, and expansion. After 3 years, 74.1% of the units had survived (66.7% H. wrightii and 88.9% T. testudinum ) with 12 H. wrightii units having expanded substantially beyond the bounds of the original PU, merging with adjacent units to form spatially continuous patches of seagrass. High survival rates for T. testudinum should be interpreted in light of concomitant declines in density and lack of significant expansion after 3 years. In its tested configuration, the GUTS was a viable method for transplanting H. wrightii where donor and receiver sites were in close proximity (<2 km; a current limitation of the GUTS design used here). However, based on the reduced density and lack of significant expansion of T. testudinum that has persisted 3 years post-transplant, the GUTS cannot yet be fully recommended for transplanting this species.     

In vitro shoot and root organogenesis, plant regeneration and production of tropane alkaloids in some species of Schizanthus

A rapid in vitro propagation system leading to formation of shoots from callus, roots, and plantlets was developed for Schizanthus hookeri Gill. (Solanaceae), an endemic Chilean plant. The genus Schizanthus is of particular interest due to the presence of several tropane alkaloids. So far, in vitro propagation of species of this genus has not been reported. Propagation of S. hookeri consisted of two phases, the first one for callus initiation and shoot formation and the second for rhizogenesis and plantlet regeneration. From a single callus that rapidly increased in cell biomass (from approximately 50 mg to approximately 460 mg/culture tube [25 x 130 mm] in 60 days) in the presence of 2.69 microM NAA and 2.22 microM BA, more than 10 shoots/callus explant were formed. From the latter, approx. twenty plantlets formed after 90-110 days shoot subculture in medium devoid of growth regulators that favored root formation. Ten alkaloids ranging from simple pyrrolidine derivatives to tropane esters derived from angelic, tiglic, senecioic or methylmesaconic acids were obtained from in vitro regenerated plantlets. One of them, 3alpha-methylmesaconyloxytropane, was not previously described. The same growth conditions, as well as other growth regulator levels tested, were required to induce callus and root formation in S. grahamii Gill. Root organogenesis occurred despite a high level of BA vs. NAA used, (i.e., 4.44 microM BA and 0.54 microM NAA); however no shoot formation was achieved. In the case of S. tricolor Grau et Gronbach, only callus formation was obtained in the presence of various growth regulators.     

18S-25S rDNA variation in tissue culture of some Gentiana L. species

18S-25S rDNA of intact plants and tissue cultures of G. acaulis, G. punctata and G. lutea have been investigated by using blot-hybridization. The decrease of rDNA amount was found in the callus cultures as compared with the plants. In contrast to other species, G. lutea showed intragenome heterogeneity of rRNA genes as well as qualitative rDNA changes in tissue culture, in particular appearance of altered repeats. The relationship between the peculiarities of rRNA gene structure and their rearrangements in in vitro culture was suggested.     

Effect of plant growth regulators,temperature and sucrose on shoot proliferation from the stem disc of Chinese jiaotou (<Emphasis Type="Italic">Allium chinense</Emphasis>) and in vitro bulblet formation

In vitro shoot proliferation from stem disc of Allium chinense, a vegetatively propagated plant, was investigated in this experiment. In the present study, shoots were formed directly on stem discs on a medium containing 1 mg/l N⁶-benzyladenine (BA) and 0.5 mg/lα-naphthaleneacetic acid (NAA). These shoots were further cultured on MS media supplemented with various levels of BA in combination with NAA, and new shoot clusters developed easily from the explants cultured despite considerable differences in the induction of shoot clusters with different levels of BA and NAA. The most productive combination of growth regulators proved to be 1.0 mg/l BA and 1.0 mg/l NAA, in which about 17 shoots were produced per cluster in 8 weeks culture. Most of the formed shoots were rooted 15 days after being cultured on MS media supplemented with 0.1–1.0 mg/l NAA. The survival rate of the plantlets under ex vitro conditions was 95% in pots filled with a peat: sand (2:1 v/v) mixture after two weeks. In vitro bulblet formation were strongly promoted by the high temperature of 30°C compared to that at 25, 20 and 15°C, and 12% (w/v) sucrose appeared to be optimal for bulblet development. Results from this study demonstrated that A. chinense could be in vitro propagated by using stem discs and in vitro bulblet formation could be achieved.     

Micropropagation of four Gentiana species (G. lutea, G. cruciata, G. purpurea and G. acaulis)

The growth of axillary shoots was initiated on nodal stem segments, excised from aseptically grown seedlings of Gentiana acaulis L., G. cruciata L., G. lutea L. and G. purpurea L. In later subcultures, a basal callus tissue developed on the shoots, giving rise to de novo formed buds. Optimum benzyladenine and indoleacetic acid combinations for shoot development were established. They were slightly different in the four species. From 35-70% of shoots rooted spontaneously, except in G. lutea, in which adventitious roots were induced by applying naphthaleneacetic acid. It was conduded that the four Gentiana species were amenable to propagation in vitro. This revised version was published online in June 2006 with corrections to the Cover Date.     

Survival dynamics of reef coral larvae with special consideration of larval size and the genus Acropora

Temporal dynamics of larval survival were examined in vitro in four broadcast-spawning reef coral species, Acropora hyacinthus, A. japonica, A. solitaryensis, and Goniastrea pectinata. Larval size was treated as an important characteristic that may relate to larval lifespan. Two patterns were observed in larval survival dynamics between the three Acropora species (mean initial larval size; 0.05-0.08 mm(3)) and G. pectinata (0.02 mm(3)), based on the timing of a sharp drop in larval survival rates (ca. > 50% reduction over a 1-2 week period). Consequently, the majority of larvae of the three Acropora species had a lifespan of less than 2-3 weeks, whereas those of G. pectinata were extended a further 2-3 weeks despite the smaller larval size. No significant relationship was detected between the initial larval size and larval lifespan in any of the four reef coral species. These results suggest that (1) larval dispersal of spawning Acropora species may be on a more local scale than that of G. pectinata and most other reef coral species previously reported, and (2) larval size is not a good estimator of larval lifespan in reef coral species.     

Effects of temperature,photoperiod and culture vessel size on adventitious shoot production of in vitro propagated <Emphasis Type="Italic">Huernia hystrix</Emphasis>

High production costs due to low growth rate in vitro and high labour costs are among factors limiting commercial application of micropropagation techniques. The low growth rate could be due to unfavourable or sub-optimal environmental and chemical conditions of the cultures. The effects of temperature, photoperiod and culture vessel size were investigated on adventitious shoot production of Huernia hystrix. There were significant increases in shoot proliferation with increased temperature in cultures maintained under a 16 h photoperiod. Slow growth observed at low temperatures (15 and 20°C) offers a potential strategy for cost-effective in vitro storage of H. hystrix germplasm. The maximum adventitious shoots produced per explant and percentage of explants producing shoots (4.2 ± 0.74 and 94% respectively) were observed in cultures maintained at 35°C, the optimum temperature for photosynthesis in plants possessing crassulacean acid metabolism (CAM). The nocturnal accumulation of organic acids in cultures incubated under a 16 h photoperiod further suggest the presence of CAM in this species. On the other hand, cultures kept under continuous light appear to shift to a C-3 photosynthetic pathway. There was a significant decrease in fresh weight of adventitious shoots regenerated per explant as temperature increased. The use of larger culture vessels further increased the shoot proliferation to 5.6 shoots per explant with a potential production of 3,429 shoots per m² in the growth room compared to 2,750 shoots per m² using culture tubes.     

Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production

This study for the first time demonstrates single bead alginate encapsulation and conversion (multiple shootlets rejuvenation) from adventitious shoot buds (AB) of Albizia lebbeck (L.) Benth. Internodal (IN) segments isolated from field grown 1-year-old plant of A. lebbeck were used for AB induction under in vitro conditions. IN segments incubated on MS medium augmented with 10.0 µM BA exhibited highest adventitious shoot bud induction frequencies (76 %) on all over the surface after 10 weeks of culture. Induced AB were detached from in vitro proliferating cultures and used for encapsulation as an explant to produce large number of synseeds (07–08) from a single IN explant. Four to five AB were encapsulated in a single calcium alginate bead to manage mass propagation, interim storage and germplasm sharing. The finer gel matrix for encapsulation was attained using 3.0 % sodium alginate and 100 mM calcium chloride. Highest percentage of shoot emergence and multiplication (75 %) from synseed was obtained on MS + 10.0 µM BA + 1.0 µM NAA (RM) after 10 weeks of culture. Encapsulated adventitious buds stored at 4 °C for 1–8 weeks (2 months) too showed thriving shoot emergence (68 %) and multiplication in encapsulated AB and development into complete plantlets when returned to RM. Hence, 4–5 encapsulated AB stored at 4 °C, when cultured back to RM also showed shoot induction resulting in up to 10 shoots per synseeds after 10 weeks of culture. Healthy root formation (½ MS + 2.0 µM IBA) and acclimatization were optimized by using previously standardized protocol (Perveen et al. in J For Res 22:47–52, 2011). Genetic stability of synseed-derived plantlets acclimatized under ex vitro was assessed and compared with mother plant using inter-simple sequence repeats (ISSR) analysis. The synthetic seeds have the achievability of being a substitute planting material for the forestry sector in future, especially for the multipurpose plant species.     

Culture of meristem tips and micropropagation of 12 commercial clones of poplar in vitro

Twelve commercial clones of poplar were cultured in vitro from meristem lips (0.3–0.5 mm diameter), shoot tips (4–6 mm long) and nodal segments (5–10 mm long). Shoot-producing cultures were obtained from 4, 32 and 70% of meristem lips, shoot tips and nodal segments within 12, 6 and 4 weeks, respectively. The genotype of cultures had a greater influence on development of shoot-producing cultures than medium composition. Cultivars Max/Ber and Oxford had the highest rates of establishment in culture and subsequent shoot proliferation, while P. tacamahaca, P. trichocarpa and cv. Robusta exhibited very low rates of establishment and low vigor in vitro. Shoot tip development was best on agar-solidified medium whereas liquid medium resulted in vitrification. Higher rates of axillary shoot production from established cultures were obtained with benzyladeninc or zeutin than with 2-isopen-tenyladenine. deducting the benzyladenine concentration from 4,4 to 1.1 μ M , increased the production of elongated shoots suitable for rooting.     

Factors affecting micropropagation and acclimatization of an elite clone of Eucalyptus tereticornis Sm.

Several factors influencing micropropagation of a selected elite clone of Eucalyptus tereticornis Sm. were investigated. Amongst different cytokinins tested, 6-benzyleadenine proved to be the most effective cytokinin for shoot multiplication and elongation. The initial size of the shoot clump (inoculum) also influenced shoot multiplication and elongation. The number of shoots proliferated per culture vessel were significantly higher (342 shoots per culture vessel) when larger shoot clumps (15?C20 shoots) were inoculated, compared to smaller shoot clumps (4?C5 shoots), which resulted in a reduced shoot proliferation rates (245 shoots per culture vessel). However, the number of elongated shoots (65 per culture vessel) and shoot length (5.23?cm) were higher in cultures which were inoculated with smaller shoot clumps in comparison to those cultures which were inoculated with larger shoot clumps (54 shoots per culture vessel with shoot length of 4.17?cm). The maximum number of rooted shoots (80.7?%) was obtained on one fourth-strength MS medium supplemented with 5.0???M indolebutyric acid. The number of shoots proliferated, elongated, rooting frequency, and subsequent survival of plants after acclimatization were higher in cultures incubated under photosynthetically active radiation (PAR) compared to those incubated under cool fluorescent lights (CFL). Osmotic potential of the sap and chlorophyll content of cultures incubated under PAR were also higher than those incubated under CFL. Following transfer of plants to soil, inoculation with a suspension of Bacillus subtilis (plant growth-promoting bacterium) increased the survival rate of plants by 10?%, yielding successful transfer of 84?% of plants. Random amplified polymorphic DNA and inter simple sequence repeat analyses indicated a high level of clonal uniformity amongst regenerated plants and also with that of the mother plant.     

Efficient shoot regeneration of Brassica campestris using cotyledon explants cultured in vitro

Summary A system has been developed for efficient regeneration of shoots from Brassica campestris in vitro. Using 4-day old cotyledons with petioles as expiants and a combination of BA and NAA in the regeneration media, up to 70% of expiants produced shoots after 2 weeks in culture. The optimal conditions for regeneration were found to include a BA concentration of 2mgL^–1 and NAA concentration of 1mgL^–1. Light intensity had a profound effect on regeneration potential. The use of silver ions as an inhibitor of ethylene action reduced regeneration rates in this system. Rooting occured simultaneously with shoot formation on these media and the resultant shoots could be rooted readily on minimal medium. The genotype dependency was investigated and indicated that this method would be widely applicable to B. campestris cultivars. Regeneration of one cultivar, a high erucic acid type (R-500), was inefficient in the system described here. Histological studies indicated the development of multiple shoot primordia from the petiolar cut ends of the expiants after the initiation of meristematic activity in the cells about 100m from the cut site within 2 days of culture initiation. The system described is compatible with previously reported Agrobacterium — mediated transformation protocols involving cotyledonary petioles.     

Cuphea wrightii thioesterases have unexpected broad specificities on saturated fatty acids

Cuphea wrightii A. Gray is an herbaceous annual that accumulates 30% caprate (10:0) and 54% laurate (12:0) in seed storage lipids. We investigated the role of acyl-acyl carrier protein (ACP) thioesterases (TE) in acyl chain-length regulation in C. wrightii. Two embryo-derived cDNAs, encoding the TEs Cw FatB1 and Cw FatB2, were isolated. Both proteins were detected in developing embryos and mature seeds but not in other tissues, suggesting involvement in seed oil synthesis. Although expected to be 10:0/12:0-ACP-specific, these genes produced a broad range of fatty acids (12:0, 14:0, and 16:0) in transgenic Arabidopsis with the greatest accumulation at 14:0. Cw FatB2 transformants also accumulated small amounts of 10:0. Because C. wrightii accumulates only ca. 5% 14:0 and ca. 2% 16:0, we tested the possibility that gene dosage effects might significantly alter the overall kinetics of the pathway. Phenotypic comparisons of progeny segregating for the transgenes individually and in a hybrid population demonstrated that increased enzyme pools in vivo had a minor effect on diverting fatty acid production to shorter chains. We propose that Cw FatB1 and Cw FatB2 may be necessary but not sufficient determinants of the C. wrightii phenotype.     

Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands

Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.     

Chromosomal variability in a tissue culture of rare species of the genus <Emphasis Type="Italic">Gentiana</Emphasis> L.

A cytogenetic analysis of plants and tissue cultures of Gentiana lutea, G. punctata, and G. acaulis is performed. It is discovered that in vitro culturing leads to changes in the chromosome number in the calli of the species. The species specificity of the variability of the genomes of the cultured cells is demonstrated. It is established that the cytogenetic structure of a tissue culture depends on the genotype of the initial plants. It is explained that in callus tissue of Gentiana (other than an in vitro culture of G. punctata, derived from a plant of the Breskul population), diploid cells and cells with near-diploid chromosome complements constitute the modal class.     

Potential of bacterial indoleacetic acid to induce adventitious shoots in plant tissue culture

AIMS: The main aim of this study was to investigate the possible role of indoleacetic acid (IAA) from bacteria to induce in vitro adventitious shoots in internodal explants of Brassica oleracea L. METHODS AND RESULTS: Culture supernatant of Halomonas sp. RE1 and Halomonas sp. HT1 that contain 21 and 40 microg ml(-1) IAA, respectively, was used to supplement Murashige and Skoog (MS) medium. Two combinations that were supplemented with bacterial supernatant (BS) are MS + BS and MS + BS + 10%CW (coconut water) while basal MS medium was used as control. The amounts of BS used in this experiment were 50, 100, 150 and 200 microl in 5 ml MS medium in each combination. In vitro-grown internodal explants of B. oleracea were inoculated on these media combinations and incubated in a growth chamber at 25 +/- 1 degrees C and exposed to 16-h cool fluorescent light. After 5-6 weeks of incubation adventitious shoot induction was observed in all treatments that were supplemented with BS as compared with the controls where very low response was observed. The frequency of shoot induction was high in media that were supplemented with 10%CW in the presence of bacterial auxin. CONCLUSIONS: It was concluded that IAA of microbial origin has the potential to induce adventitious shoots in internodal explants. SIGNIFICANCE AND IMPACT OF THE STUDY: IAA from bacteria can be effectively used in plant tissue culture; especially a combination of MS + BS + 10%CW is very cost-effective as compared with synthetic phytohormones for in vitro studies.     

Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron

An efficient in vitro method for multiple shoot bud induction and regeneration has been developed in Artemisia annua L. using leaf and stem explants in various concentrations and combinations of plant growth regulators to evaluate the frequency of regeneration. The sources of explants as well as plant growth regulators in the medium were found to influence the multiple shoot induction. The result shows that the stem segment cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg/l thidiazuron (TDZ) gave a perfect shoot formation (100%) and good shoot multiplication (57 shoots/explant) after 2 weeks of culture. Healthy regenerated shoots were elongated and rooted in MS medium without hormones. The artemisinin content in plants regenerated from stem explants using 0.1 mg/l TDZ was (3.36 +/- 0.36) microg/mg dry weight and two-fold higher than that of in vitro grown plants of the same age [(1.73 -/+ 0.23) microg/mg DW]. This system exhibited a potential for a rapid propagation of shoots from the stem explant and makes it possible to develop a clonal propagation of A. annua.     

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20 min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2 M sucrose solution for 20 min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10 days of plating and developed shoots within 3 weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.