Factors affecting haploid induction through in vitro gynogenesis in summer squash (Cucurbita pepo L.)

Haploid production using in vitro ovule cultures has long been recognized as an important tool to produce haploid and homozygous double-haploid plants for genetic studies and plant breeding programs. In the present study, four experiments were carried out to study the influence of genotype, position of female flowers on plant stem, temperature and sucrose concentration on the in vitro gynogenesis induction of squash. (1) Ovules of 12 genotypes were excised from female flowers, 1 day before anthesis, and cultured onto MS medium containing 3% sucrose and 1 mg l⁻¹ from each of kinetin and 2,4-D (2,4- dichlorophenoxy acetic acid). Differences in response among genotypes were demonstrated. Raad F₁ showed the highest percentage of responding ovules and number of plantlets per dish with 48.8% and 15 plants, respectively. The results revealed that genotype is a key factor influencing the in vitro gynogenesis in squash. (2) Ovules were excised from first, second and third female flower of two hybrids (Giad and Raad) and cultured onto the mentioned above medium. The highest percentage of responding ovules and number of plantlets per dish were obtained from ovules excised from the second female flower on the plant stem. (3) Effect of temperature (4 and 32 °C) for 0, 4, 7 and 12 days on the ovule culture of Queen F₁ was studied. Ovules incubated at 4 or 32 °C for 4 days produced a better embryogenic response. (4) Three sucrose concentrations (30, 60 and 90 g l⁻¹) were tested with the ovule cultures of the local cultivar (Eskandrani). Differences among sucrose concentrations were statistically significant and ovules cultured on the MS medium containing 30 g l⁻¹ produced the best result. MS medium containing 90 g l⁻¹ did not produce gynogenic ovules.     

Increased regeneration capacity in spinach lines obtained by in vitro self-fertilisation

Somatic embryos (SEs) were induced from apical sections of the lateral roots of spinach seedlings (1 cm), which were cultivated on solid Murashige and Skoog (MS) medium with 20 μM α-naphthaleneacetic acid and 5 μM gibberellic acid. Apical shoots of the same lines were isolated and cultivated on plant growth regulator-free medium. The regeneration capacities of seedlings randomly chosen from a population were quite low and variable, and only 4 out of 30 lines responded at the frequency of 85–100%, with 6.96–9.96 SEs per explant and up to 347 SEs per seedling over a 12-week period. These SEs were isolated and maintained on medium with 5 μM kinetin. Plants derived from seedlings’ apical shoot and SEs self-fertilised in vitro and set seeds, and these seedlings (S₁) were used to induce regeneration. Similarly, S₂–S₄ seedlings were obtained and the regeneration capacities of 23 S₁, 23 S₂, 17 S₃ and 5 S₄-seedlings were compared to parental lines. Of these, four S₃ and S₄ lines with extremely high regeneration capacities were selected. These lines exhibited 78–139 fold higher embryo-forming capacities than the mother plant, and produced 38.9–68.3 SEs per explant and 1339–2181 SEs per seedling during the same time period. In addition, the process of somatic embryogenesis began 2–4 weeks earlier in these lines, and root explants taken from SE-derived plants of these lines retained high and stable regeneration capacities, and therefore may be ideal material for genetic transformation.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

Ethylene influences development and flowering of Ptilotus spp. in vitro and ex vitro

The genus Ptilotus has immense potential for ornamental horticulture but its commercial development has been hindered by propagation limitations. Poor seed quality and germination are reported. Cutting propagation is limited by cutting supply as the juvenile phase of Ptilotus is short. Micropropagation has been used in an attempt to overcome these difficulties but explants become floral in vitro and this causes plantlets to elongate. Ethephon has been used to control flowering of stock plants of many ornamental species. This study investigated the effect of ethephon applied to young (3-week-old, deflasked from tissue culture) and mature (1-year-old) Ptilotus plants in a greenhouse. A system of applying gaseous ethylene at 0, 100, 200 and 300 mg l⁻¹ to the headspace of in vitro plantlets in glass jars was developed and the response of in vitro plantlets to ethylene studied. One-year-old Ptilotus plants were treated with 500 mg l⁻¹ ethephon 2 days before pruning or 1 or 2 weeks after pruning. Ethephon application 2 days before pruning decreased the number of inflorescences and increased the number of shoots (compared to the control) but was phytotoxic. Ethephon applications of 150 or 300 mg l⁻¹ applied weekly or fortnightly to 3-week-old plants deflasked from tissue culture reduced plant height and number of inflorescences and at low concentrations increased the number of new shoots. A fortnightly application at 150 mg l⁻¹ is recommended. Previous reports on the effects of ethylene on inflorescence production on plantlets in vitro are limited. Our study showed that exposure of in vitro plantlets of P. nobilis to ethylene gas at 100 mg l⁻¹ for 1 h significantly increased the number of shoots and plant height but this did not occur for plantlets of P. spicatus. Plantlets of P. spicatus exposed to transient ethylene at 200 and 300 mg l⁻¹ showed significantly greater rooting (52.4%) than the control (13.6%).     

Susceptibility of grapevine viruses to thermotherapy on in vitro collection of Kober 5BB

A number of virus elimination protocols have been developed to obtain healthy plants but their success rates have been variable due to the strong virus–host relationship, treatment performances and experimental condition. An in vitro collection of plants of Vitis berlandieri × Vitis riparia Kober 5BB single-infected by Grapevine vitivirus A (GVA), Grapevine fanleaf nepovirus (GFLV), Grapevine fleck maculavirus (GFkV), Grapevine leafroll ampelovirus 1 (GLRaV-1) and Grapevine leafroll ampelovirus 3 (GLRaV-3) was established to carry out thermotherapy treatments. The experimental system allowed to control the effects of host genotype by using the same genetic background, and the environmental condition such as relative humidity, soil and temperature conditions. Trials were conducted in a growth chamber set at 37 ± 0.5 °C for 48 days using in vitro cultures for all infections. ELISA and RT-PCR were used to check sanitary conditions. Results showed complete eradication of GFLV and no effect against GFkV. Different sanitation rates were obtained for other phloematic viruses: 70.2% for GVA, 25.1% for GFLaV-1 and 24.7% for GLRaV-3. The results on phloematic viruses showed a gradient of elimination efficiency which could not be completely explained with the location of viral particles that seems to be only one of the several factors affecting virus susceptibility to heat stress. Moreover, the advantages of this experimental system provide the opportunity to obtain a greater understanding of the thermotherapeutic effects on phloematic viruses.     

Regeneration and characterization of plants derived from leaf in vitro culture of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars

In the current work attempts were made to investigate culture of leaf explants derived from in vitro seedlings of two sweet orange (Citrus sinensis (L.) Osbeck) cultivars, Bingtangcheng and Valencia. Effects of several factors, including culture medium, lighting condition, explant age and genotype on regeneration response were examined based on three parameters, percentage of explants producing shoots, mean number of shoots per explant and shoot forming capacity. Culture of the explants on shoot-inducing media (SIM) composed of MT salts supplemented with different growth regulators gave rise to disparate shoot regeneration, in which SIM₁ (MT + 0.5 mg L⁻¹ BA + 0.5 mg L⁻¹ Kinetin + 0.1 mg L⁻¹ NAA + 3% sucrose + 0.8% agar, pH 5.8) was shown to be the most effective medium for direct induction of shoots from leaf explants. Highly significant difference in the response of shoot bud regeneration was noted between the two cultivars, with Bingtangcheng being more responsive than Valencia. Culture of explants from fully developed leaves led to better shoot regeneration capacity in comparison to undeveloped ones. However, the two lighting conditions used herein did not cause significant difference in shoot regeneration. Phenotypic observation and randomly amplified polymorphic DNA (RAPD) analysis confirmed that all the regenerated plants from both genotypes were genetically identical to their donor plants, suggesting absence of detectable genetic variation in the regenerated plants. The data presented here demonstrated that direct initiation of plants from leaf explants has been successfully accomplished. To our knowledge, this is the first report on direct regeneration of shoots from leaf explants in Citrus, which will provide an alternative source for citrus genetic manipulation in the future.     

Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification

In this study in vitro shoot tips of a Sicilian genotype of Limonium serotinum were successfully cryopreserved using the droplet-vitrification technique. Growth recovery of cryopreserved shoot tips was possible only when samples were pretreated for 16 h in liquid medium with 0.3 M sucrose, then for 5 h in liquid medium with 0.7 M sucrose before performing the cryopreservation protocol. Optimal conditions included treatment for 20 min in a loading solution containing 1.9 M glycerol + 0.5 M sucrose, treatment with vitrification solution B5 (glycerol 40.0%, sucrose 40.0%, w/v) for 60 and 90 min or vitrification solution A9 (glycerol 30.0%, dimethylsulfoxide 20.0%, ethylene glycol 20.0%, sucrose 15.0%) for 20 min, rapid cooling in minute droplets of vitrification solution, rapid rewarming by immersion for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 37% recovery of cryopreserved shoot tips was achieved. Regrowth of cryopreserved samples was slow but always direct, without callus formation.     

Direct regeneration of plants derived from in vitro cultured shoot tips and leaves of three Lysimachia species

The regenerability of three ornamental species—Lysimachia christinae, Lysimachia rubinervis and Lysimachia nummularia ‘Aurea’, were investigated using in vitro leaves and shoot tips. 6-Benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA) added to Murashige and Skoog (MS) medium were tested for their effect on organogenesis. On the medium, shoot regeneration occurred directly without callus formation. In these species, L. christinae developed the highest regeneration rate and numbers of shoots/explant from shoot tips (100%, 12.25) and leaf bases (100%, 13.01) on the MS medium containing 3.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. For L. rubinervis, the highest shoot induction rate and number of shoots/explant were obtained from shoot tip (100%, 16.87–17.20) on the MS medium with 0.1 mg l⁻¹ NAA and 3.0–5.0 mg l⁻¹ BAP. L. nummularia ‘Aurea’, however, showed the highest regeneration rate and number of shoots/explant (100%, 12.73) from leaf bases on MS medium supplemented with 1.0 mg l⁻¹ BAP and 0.1 mg l⁻¹ NAA. All in vitro shoots rooted well on half macronutrient MS medium containing 0.1 mg l⁻¹ NAA. After acclimatization, transplanted plantlets grew normally and flowered in the field.     

Screening Capsicum species of different origins for high temperature tolerance by in vitro pollen germination and pollen tube length

Successful fruit set depends on several reproductive processes including pollen germination and tube growth processes. An experiment was conducted to determine the effects of temperature on pollen germination characteristics and to identify species/genotypic differences in Capsicum using the cumulative temperature response index (CTRI) concept. Pollen was collected from plants of seven genotypes from five Capsicum species, adapted to various parts of the world and grown outdoors in large pots. The pollen was subjected to in vitro temperatures ranging from 15 to 50 °C at 5 °C intervals. Pollen germination and tube lengths were recorded for all species after 24 h of incubation at the respective treatments. Species/genotypes differed significantly for in vitro pollen germination percentage and pollen tube length with mean values of 78% and 734 μm, respectively. The mean cardinal temperatures (T_min, T_opt, and T_max) averaged over genotypes, were 15.2, 30.7, and 41.8 °C for pollen germination and 12.2, 31.2, and 40.4 °C for pollen tube growth. The CTRI of each species/genotype calculated as the sum of eight relative individual stress response values, such as maximum pollen germination, maximum pollen tube length; T_min, T_opt, and T_max temperatures of pollen germination, and pollen tube lengths, identified species tolerance to high temperatures. Capsicum annum cv. Mex Serrano from Mexico was identified as tolerant, C. chacoense cv. 1312 and C. spp. cv. Cobanero from Argentina and Guatemala, respectively as intermediate and C. frutescens cv. Early Spring Giant from China, C. annum cv. Long Green from South Korea, C. spp. cv. NM89C130 and C. pubescens cv. 90002 from Guatemala as sensitive to high temperatures. The tolerant species/genotypes can be used in breeding programs to develop new genotypes that can withstand high temperature conditions both in the present climate and particularly in a future warmer climate.     

Genetic diversity and population's structure in Tunisian strawberry tree (Arbutus unedo L.)

A growing interest in the use of the Strawberry tree (Arbutus unedo L., Ericaceae) has been recently reported for industrial, pharmaceutical and chemical fields. However, the bulk material comes from natural populations because of the lack of selection of interesting cultivars. In Tunisia, A. unedo populations are severely destroyed due to deforestation and over-collecting. The species occurs in small scattered populations decreasing progressively in size. Yet, no conservation or improvement programs are attempted to preserve and promote the potential value of this resource. In this work, we assessed the genetic diversity of nine Tunisian populations of A. unedo L. from different bioclimates, using 65 polymorphic RAPD loci. The analysis of the genetic variation within and among populations is primordial to elaborate conservation and improvement programs. A low genetic diversity within a population estimated by both Nei's (He) and Shannon's diversity (H′) indices (0.155 < He < 0.248; 0.229 < H′ < 0.364) was observed due to genetic drift and selfing. At the species level, the amount of the within population variation estimated by Shannon's index (H_POP/H_SP = 0.686) and the molecular variance (80.67%) was higher than that among populations. A moderate genetic differentiation among populations (Φ_ST = 0.193 and G_ST = 0.314) which could be attributed to the long seed distance dispersal was detected. The UPGMA dendrogram based on Φ_ST values showed three clusters each including populations without relationship to bioclimatic or geographical origin indicating that differentiation occurs at a local space scale. The in situ protection measures should be made appropriately according to a population within bioclimates. The ex situ conservation and the selection of genotypes should involve extensive collection of seeds or cuttings from the within populations rather than among them.     

Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.     

Genetic transformation of Prunus domestica L. using the hpt gene coding for hygromycin resistance as the selectable marker

The study and development of transformation technology with new selection schemes is important for various fundamental studies and for crop trait improvement via genetic engineering. Here we have shown that hygromycin resistance is an effective system for plum genetic transformation. Embryonic axes of mature seeds were co-cultivated with Agrobacterium strain LBA4404 containing the pC1381 plasmid carrying the hygromycin phosphotranferase gene (hpt) and β-glucuronidase (GUS) gene or with strain EHA105 containing the plasmid pC1301 carrying the same marker and reporter genes. The latter strain containing a pC2301 plasmid carrying the neomycin phosphotransferase gene (nptII) gene was used as a control. Infected explants were placed on shoot induction medium containing either 5 mg L⁻¹ hygromycin or 75 mg L⁻¹ kanamycin for selection. Green shoots developed from the explants under hygromycin pressure. These shoots showed continued and vigorous growth and development upon transfer onto fresh hygromycin medium. PCR using hpt sequence primers, and Southern blot analysis using a probe from the hpt gene, confirmed the presence of the transgenes and their stable integration in regenerated plants. Full transgenic plants were obtained in a greenhouse. Hygromycin selection was very effective and no escapes were observed. The study demonstrated that hygromycin resistance can be used as an effective selectable marker for plum transformation. The new system developed here is important and useful for multiple gene transformation in plum.     

Effect of cold cathode fluorescent lamps on growth of Gerbera jamesonii plantlets in vitro

The goal of this study was to evaluate the effect of cold cathode fluorescent lamps (CCFLs) on the growth of Gerbera jamesonii var. ‘Rui Kou’ plantlets in vitro in six different light quality ratios: 100% red CCFL (R), 80% R + 20% blue CCFL (B), 70% R + 30% B, 60% R + 40% B, 100% B and white CCFLs (W). Control radiation was provided by conventional heat-generating plant growth fluorescent lamps (PGFLs). Plantlets under CCFLs showed better plantlet height, SPAD value (i.e., leaf chlorophyll content) and root activity (as assessed by root dehydrogenase activity) than those growing under PGFLs while all other growth parameters were comparable with plants under conventional lighting systems.     

Correlation of saponin content and Fusarium resistance in hybrids from different ploidy levels of Lilium Oriental

Lilium Oriental is of great commercial value, but large losses in production can result from its susceptibility to diseases caused by Fusarium ssp. Here we report a mutant of Lilium Oriental resistant to Fusarium, Cai-74, which was generated from crossing tetraploid (from ‘Star fighter’) and diploid (‘Cor. Amore’ × ‘Acapulco’) plants. Hybrid progeny were screened via inoculating Fusarium oxysporum into tissue cultured plantlets in a greenhouse. Although Cai-74 had a saponin content of 3.81 mg g⁻¹, which is much higher than its parents, the highly Fusarium-resistant wild species Lilium dauricum had the highest content of 4.11 mg g⁻¹ among the tested genotypes. Cai-74 had a 3A karyotype rather than 3B found in normal plants of Lilium Oriental. Taken together, our results suggest that Cai-74 bears a chromosomal variation in its structure, and that the saponin content in the lily scales correlate with the Fusarium resistance of Lilium Oriental. Cai-74 could be used as a gene resource for breeding Fusarium-resistant cultivars of Lilium Oriental.     

High frequency early flowering from in vitro seedlings of Dendrobium nobile

Dendrobium nobile Lindl. is a popular temperate Chinese orchid commonly marketed as a traditional medicinal plant. Seedlings of Dendrobium nobile Lindl. produced floral buds (33.3–34.8%) precociously on a defined basal medium (1/2 MS) containing paclobutrazol (PP₃₃₃) at 0.5 mg L⁻¹ or thidiazuron (TDZ) at 0.1 mg L⁻¹ within 4 months of culturing. The frequency of floral buds formation can be further increased to 95.6% by growing seedlings in a PN (PP₃₃₃ 0.3 mg L⁻¹ + NAA 0.5 mg L⁻¹)-containing medium followed by transfer onto 1/2 MS medium with PP₃₃₃ and TDZ (PP₃₃₃ + TDZ). However, flower developed was deformed under 25 °C but it developed fully when grown in a lower temperature regime (23 °C/18 °C, light/dark) for 45 days. Under optimal condition, in vitro flowering was observed about 6 months after seed sowing.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Fingerprinting of three typical macrosperma Italian lentil (Lens culinaris Medik.) landraces using fluorescence-based AFLP markers

Italian lentil landraces are principally cultivated for self or local consumption. Most of them are disappearing, particularly macrosperma types by being less required by the market. A pre-requisite for the conservation and the efficient use of genetic resources is the better understanding of the extent and the distribution of the existing genetic variation, useful for future breeding programmes. Our study was undertaken to analyse and quantify the genetic diversity within and among three macrosperma Italian lentil landraces (Onano, Altamura and Villalba), using fluorescent AFLP markers. AFLP markers generated information to differentiate among closely related genotypes and group within the same cluster individuals belonging to the same landrace. The total genetic diversity (H_T), the genetic diversity within population (H_S) and the extent of differentiation between populations (D_ST) were 0.198, 0.155 and 0.043, respectively. The fixation index (G_ST = 0.219) showed that about 78% of the observed total genetic variation can be attributed to within population differences and around 22% is due to differences among populations. The gene flow estimate (N_m = 1.774) and the mean genetic distance value (0.077) suggested narrow genetic base among the analysed populations, confirming the tendency of Italian lentil landraces to group together. The present study showed that fluorescence-based AFLP technique is a biotechnological tool that can provide significant insights for research in genetic diversity of lentil landraces and their subsequent conservation and utilization in breeding programs.     

Plant regeneration from in vitro cultured leaves of Lanzhou lily (Lilium davidii var. unicolor)

Lanzhou lily (Lilium davidii var. unicolor) is one of the best lilies which are edible in China but the efficient shoot regeneration system has not been developed. The purpose of the present study is to establish an efficient and reproducible protocol for induction of shoots in vitro from L. davidii var. unicolor leaves. Shoot regeneration from in vitro cultured leaves of L. davidii var. unicolor was tested on the 26 media based on NN [Nitsch, J.P., Nitsch, C., 1969. Haploid plants from pollen grains. Science 163, 85–87] basal medium, containing different concentrations of thidiazuron (TDZ) in combination with different concentrations of α-naphthaleneacetic acid (NAA). Shoot organogenesis occurred directly from the leaves without forming callus. Shoot regeneration mainly occurred from the cuts across the midvein and the base of the leaf explants. The highest frequency of regeneration (93.3%) and the largest number of shoots per leaf (3.83) were obtained on NN basal medium supplemented with 0.5 mg l⁻¹ TDZ and 1.0 mg l⁻¹ NAA. All the regenerated shoots formed complete plantlets on half-strength MS [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497] basal medium containing 0.1–0.5 mg l⁻¹ indole-3-butyric acid (IBA) with in 30 days, and 92% of the regenerated plantlets survived in the soil. This study will be useful for Agrobacterium-mediated transformation and exploitation of somaclonal variation of Lanzhou lily.