In vitro bulb production fromAllium spp.

Summary A procedure for bulb formation from onion, garlic, and shallot explants is described. Explants from cut stem bases were cultured in shoot induction medium composed of Murashige and Skoog (MS) medium with or without N⁶-benzyladenine. Shoots produced were then transferred to bulb induction medium composed of MS medium containing 5 g/liter activated charcoal and 120 g/liter sucrose under a long-day photoperiod and 28° C. Bulbs were also produced from onion and garlic directly, without passing through shoot formation, when explants were cultured in the bulb induction medium described above. Bulbs were transferred to soil without acclimatization and produced viable plants.     

Effects of explants and growth regulators in garlic callus formation and plant regeneration

We intended to evaluate the effects of different explants and growth regulators on callus induction and plant regeneration in garlic (Allium sativum L.). Furthermore, we intended to differentiate among different morphological types of callus by light microscopy and to relate them with their abilities to regenerate plants in the red-garlic cultivar 069. A factorial design with BDS—basal Dunstan and Short (1977)—medium, as a control and supplemented with 0.042, 0.42 and 4.24 μM picloram or with 0.045, 0.45 and 4.5 μM 2,4-D, in both cases with and without 4.43 μM N⁶-benzylaminopurine (BAP), was used. The cultures were grown in darkness at 25 ± 2°C and they were subcultured over a 6-month period. Basal plates and meristems were highly responsive explants, while immature umbels and root-tips were less responsive ones, as indicated by percentage of induced callus, growing callus and regenerating callus. The best response was 41% regenerating callus with 0.045 μM 2,4-D and BAP from basal plates while 57, 56 and 20% regenerating callus were obtained with 0.45 μM 2,4-D from meristems, root-tips and immature umbels, respectively. Also, these treatments showed a higher percentage of nodular and embryogenic callus (type I). Thus, it can be concluded that the use of meristems and 2,4-D will enhance callus production and quality, increase plant regeneration and allows to develop a protocol suitable for further transformation experiments in garlic.     

The effects of storage condition on starch metabolism and regeneration potentials of twin-scales and inflorescence stem explants of <Emphasis Type="Italic">Narcissus tazetta</Emphasis>

Summary The natural propagation rate of Narcissus is very slow. In vitro micropropagation of Narcissus is more efficient than conventional propagation for rapidly building up aseptic stocks of varieties, especially for the establishment of new cvs. and the production of pathogen-free stock material. In the present study, Narcissus tazetta cv. ‘Ziva’ bulbs were used as the source of mother plants. The bulbs were kept at 30°C in a dark chamber until the start of the experiments. Prior to explant preparation, the bulbs were subjected to a cold treatment at 15°C in the dark for 6 wk to break dormancy. Twin-scales and inflorescence stem discs were isolated from aseptic bulb parts and were used as the initial explants. The polar orientation affected the regeneration of the inflorescence stem. Storage duration at 30°C followed by cold treatment were found to affect starch levels, adenosine diphosphate glucose pyrophosphorylase (AGPase) activities, and regeneration potentials. Starch levels were reduced significantly during a 10 mo. storage period at 30°C in both twin-scales and inflorescence stem disc explants. Regeneration was followed by an efficient acclimatization system with 98–99% survival. More than 500 highly uniform young bulbs were propagated from one mother bulb in a 12 mo. period.     

Control of pattern of regenerant differentiation and plantlet production from leaflet segments of <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss. (neem)

A process with controlled pattern of regenerant differentiation from leaflet segments leading to production of cloned plants of a 40-year-old tree of Azadirachta indica was developed. A two-step procedure was adopted for containing intervening callusing during regenerant differentiation using modified Murashige and Skoog (MS) medium, where in the first step the explants were subjected to pulse treatments having higher concentration of 6-benzylaminopurine (BAP), while in the second step they were cultured in one-tenth of the initial concentrations of BAP. In the present case, simultaneous differentiation of two types of morphogenetic structures, that is, shoot buds and the meristematic nodules was observed. However, differentiation of higher number of desirable regenerants—the shoot buds and a few meristematic nodules, rather than vice-versa could be controlled by increasing both, the concentration of BAP in pulse treatment and the duration of pulse treatment. In the optimum treatment, where the explants were exposed to 8.88 μM BAP and 81.43 μM adenine hemisulphate for 5 days followed by their transfer to 0.88 μM BAP and 81.43 μM adenine hemisulphate, on an average, 17.4 shoot buds and only 1.6 meristematic nodules were formed from a leaflet. On subculturing, the shoot buds developed into shoots, whereas the meristematic nodules produced three kinds of organized structures that too in varied proportions. Multiplication of shoots was sustained in proliferation medium supplemented with 1.11 μM BAP, 1.43 μM indole-3-acetic acid (IAA) and 135.72 μM adenine hemisulphate. The isolated shoots were rooted and complete plantlets were transferred to potted soil with 100% survival.     

Plant regeneration from seedling explants of green bean (Phaseolus vulgaris L) via organogenesis

Green bean (Phaseolus vulgaris L.) plants were regenerated from 3-day old seedling explants via organogenesis. The explants contained a cotyledon and a small portion (2–3 mm) of embryonic axis split in half. Explants were cultured on a defined medium containing glutamine as the sole nitrogen source. A ring of meristematic tissue was produced at the base of the axillary bud located at the cotyledonary node. The meristematic tissue was produced only if the axillary bud was present together with the cotyledon in the explant. Buds and shoots developed from the meristematic ring. Selected shoots produced roots when excised from the cluster of buds and transferred to root induction medium. Rooted shoots (plantlets) grew well and produced viable seeds when grown in the greenhouse. Histological studies revealed the origin of buds from the peripheral layers of the meristematic ring.Production of buds and shoots was a continuous process, so that new shoots could be removed from the explant for plantlet production every 10–14 days. With the cultivar Dark Red Kidney, an average of 49 buds and 8 shoots were regenerated per explant by 30 days after culture initiation. Sixty-seven percent of the shoots produced roots, and 90–95% of the plantlets survived greenhouse acclimatization to produce healthy plants.     

Optimization of in vitro multiple shoot clump induction and plantlet regeneration of Kentucky bluegrass (Poa pratensis)

An efficient protocol for Kentucky bluegrass (Poa pratensis L.) in vitro culture was established using shoot apices of seedlings as explants. The optimal procedure of this protocol for majority of the genotypes was that meristematic cell clumps and small calluses were firstly induced from the bases of explants on initial culture medium supplemented with 0.9 μM 2,4-d and 8.9 μM 6-BA for 20 d, then were separated and transferred to shoot clumps induction medium containing 8.9 μM 6-BA for the formation of multiple shoot clumps. The percentage of multiple shoot clumps and numbers of shoots per clump were deeply related with the combinations of different plant growth regulators, duration of initial culture, the intensity of illumination and genotypes. Histological observation of the induced explants revealed that the meristematic cell clumps were produced from repeated division of the cortical cells and original meristematic primodium cells of explants, and the multiple shoots were formed via organogenesis pathway in the meristematic cell regions of cultures on shoot clumps induction medium. In this study, plantlets were efficiently regenerated on large scale from seven cultivars of Kentucky bluegrass. Hence the meristematic cell clumps and small calluses in this protocol could be considered good targets for genetic transformation of Kentucky bluegrass.     

A simple shoot multiplication procedure using internode explants,and its application for particle bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in watercress

A shoot multiplication system derived from internode explants was investigated with the aim of improving genetic characteristics of watercress (Nasturtium officinale R. Br.). Internodes of ca. 1 cm excised from in vitro stock shoot culture were placed on half-strength Murashige and Skoog (MS) medium supplemented with 3 μM 2,4-dichlorophenoxyacetic acid as a pre-treatment. Laser scanning microscopy indicated clearly that the first sign of meristematic cell division could be seen after 1–2 days of pre-culture, and meristematic tissues multiplied along the vascular cambium of the internode segment during 7 days of culture. Multiple shoots could be obtained from more than 90% of the pre-treated explants when they were subsequently transferred to MS medium supplemented with 1 μM thidiazuron for 3 weeks. These findings indicate that pre-treatment of the internodes for 7 days promoted their capacity for organogenesis. Using this pre-treatment, frequent generation of transgenic watercress plants was achieved by adapting particle bombardment and Agrobacterium-mediated transformation techniques with a construct expressing a synthetic green florescent protein gene.     

Virus Free Garlic (Allium Sativum L.) Plants Obtained by Thermotherapy and Meristem Tip Culture

Virus infection in garlic considerably reduces yield and quality in Argentina. The production of virus free “seed” was attempted by means of thermotherapy and meristem tip culture. A hot water treatment was employed to determine the lethal temperature/time combination for clonal type (c.t.) Blanco cloves. It was established that 50°C × 20 min, 50°C × 15 min and 55°C × 5 min were the limit thermal/time combinations which garlic could withstand. Those treatments were employed followed by meristem tip culture, however, none of the successfully developed plants after culture (only 13 %) were virus-free. Hot air treatments in a growth chamber at 36°C lasting for 30, 40 and 60 days, and at 25°–32° for 30 days in a greenhouse were tested on c.t. Blanco. Cloves kept at room temperature throughout the experiment were employed as controls. In the 25°–32°C treatment, 73% of meristems produced plants and, of these, 33 % were virus free. After 30 and 40 days at 36 °C, 62 % and 67 % of the meristems developed into plantlets, of which respectively 51 % and 50 % were virus-free. Very few meristems (10 %) developed into plants when cloves had been kept at 36°C for 60 days but the resulting plantlets were all virus free. Controls produced 78 % of plants, of which 14 % were virus free. Results of hot air treatments of 36 °C for 40 days performed on c.t. Colorado, Rosado, Paraguayo, Espaol and Hilario Ascasubi were similar to those obtained with c.t. Blanco. In Espaol and Hilario Ascasubi, no virus-free plants were detected among control specimens (no thermotherapy treatment). The only virus (from up to 3 that infected the plants) that persisted in some plants after themotherapy and meristem tip culture was garlic yellow streak.     

Effects of genotype, explant source, and plant growth regulators on indirect shoot organogenesis in Dieffenbachia cultivars

The capacity for indirect shoot organogenesis of leaf and root explants of four Dieffenbachia cultivars were examined on a modified Murashige and Skoog (MS; Physiol Plant 15:473–495, 1962) medium supplemented with different plant growth regulators in 112 combinations. Callus formation was only observed from leaf explants on MS supplemented with 1–10 μM thidiazuron (TDZ) and 0.5–1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) regardless of cultivars. The combination of 5 μM TDZ and 1 μM 2,4-D resulted in the greatest callus formation frequency among the four cultivars tested. Significant differences in callus and shoot formation from leaf explants were also observed among cultivars. Cultivars Camouflage, Camille, Octopus, and Star Bright produced green nodular, brown nodular, yellow friable, and green compact calli with corresponding maximum callus formation frequencies of 96%, 62%, 54%, and 52%, respectively. A maximum of 6.7 shoots/callus was observed in cv. Camouflage, followed by cvs. Camille and Star Bright at 3.7 and 3.5, respectively. Calli of cv. Octopus displayed no capacity for shoot organogenesis. Regardless of cultivar, callus formation was not observed on root explants. Regenerated shoots were successfully acclimatized in a shaded greenhouse condition with 100% survival.     

Morphogenetic responses from protoplasts and tissue culture of Laminaria digitata (Linnaeus) J. V. Lamouroux (Laminariales, Phaeophyta): callus and thalloid-like structures regeneration

The regeneration of meristematic tissues from sporophytes of Laminaria digitata was studied by protoplast and tissue culture. Sequential treatment of explants in sterile seawater with 1% Betadine for 5 min, 1% commercial bleach for 1–2 min and 2% antibiotic treatment supplemented with 1 μM GeO₂ overnight enabled viable explants as high as 55%. Different morphogenetic responses were observed from tissue culture on media supplemented with plant growth regulators alone or in combination, mainly filamentous calluses up to 50% according to the media. Dark green compact calluses were observed on two combinations: 4 μM Pi + 2 μM N-(2-chloro-4-pyridyl)-N’-phenylurea (CPPU) and 0.04 μM Pi + 0.44 μM 6-benzylaminopurine. Thalloid-like structures comparable to adventitious buds were regenerated on medium supplemented with 4 μM Pi + 0.45 μM zeatin but at low frequency suggesting a strong genotypic effect. Friable calluses were developed from protoplasts in enriched medium with polyamines and containing 0.40 μM CPPU + 0.45 μM 2,4-dichlorophenoxyacetic acid. In order to produce protoplasts, a one-step enzymatic protocol was developed and yields reached 22 × 10⁶ protoplasts per gram of fresh weight.     

The development of a reproducible Agrobacterium tumefaciens transformation system for garlic (Allium sativum L.) and the production of transgenic garlic resistant to beet armyworm (Spodoptera exigua Hübner)

This paper describes the development of a reliable transformation system for garlic (Allium sativum L.) and its application in producing insect resistant GM garlic lines. The transformation system is based on Agrobacterium tumefaciens as a vector, using young callus derived from different callus sources: callus induced from both apical and non-apical root segments of in vitro plantlets, true garlic seeds and bulbils. Two different reporter genes were used in our garlic transformation experiments, namely the gusA gene coding for -glucuronidase and the gfp gene coding for green fluorescent protein. A total of seven independent transformed callus lines derived from different callus sources were obtained. The advantage of the system developed is the short time period needed for completion of the protocol (about 6 months) and the year-round availability of high quality callus from in vitro roots. The highest transformation frequency in a single experiment (1.47%), was obtained using garlic cv. 'Printanor'. Differences existed between cultivars in transformation frequency but were not significant. The same was found for the plasmids used in transforming garlic. Via PCR the presence of the gusA, hpt (hygromycin phosphotransferase) and gfp genes could be demonstrated in putative transformed in vitro plants. Southern hybridization showed that the reporter gene gusA and the selective gene hpt were stably integrated into the garlic genome. After transfer to the greenhouse of in vitro regenerants, transgenic garlic harbouring the gusA gene survived and grew well, whereas the gfp transgenic garlic gradually died under these conditions.Using this protocol transgenic garlic resistant to beet armyworm using the cry1Ca and H04 resistance genes from Bacillus thuringiensis were developed. Via Southern hybridization it was shown that the cry1Ca sequence was stably integrated into the garlic genome. After transfer of the transgenic in vitro garlic plants to the greenhouse, the cry1Ca plants developed normally and grew well to maturity with normal bulbs. However, all transgenic in vitro H04 garlic plants did not survive after transfer to the greenhouse. Transgenic cry1Ca garlic plants proved completely resistant to beet armyworm in a number of in vitro bio-assays. This finding will facilitate the development of new garlic cultivars resistant to beet armyworm.     

The effect of jasmonic acid, sucrose and darkness on garlic (Allium sativum L. cv. Ptujski jesenski) bulb formation in vitro

Summary The effect of sucrose, jasmonic acid (JA) and darkness on bulb formation of garlic Allium sativum L. cv. Ptujski jesenski was studied in vitro. B5 medium supplemented with 3% sucrose, 5 μM JA and 5 μM 2-isopentenyl adenine (2iP) was used for shoot induction on garlic basal plates. For bulb induction, explants with developed shoots were transferred onto media with 3% or 8% sucrose in the presence or absence of 5 μM JA. Sucrose (8%) significantly increased the percentage of shoots which formed bulbs by 86–90%, bulb diameter and the number of bulbs per basal plate. On medium supplemented with JA, the average number of bulbs per basal plate was 11.5. Growth of explants in the dark was ineffective for stimulating bulb formation. Simultaneous use of JA and sucrose can improve garlic micropropagation via bulb formation, without intermediate callus formation.     

Nucleolar vacuolation in soybean root meristematic cells during recovery after chilling

The nucleolar vacuole formation in soybean root meristematic cells from seedlings grown 3 d at temperature 25 °C (control), 3 d at temperature 25 °C and then transferred to 10 °C (chilling) for 4 d, and after recovery for 1.5, 3, 6, 12 and 24 h at 25 °C were observed on semi-thin sections. Simultaneously, autoradiographic studies with ³H-uridine on squashed preparations were carried out. During recovery of plants, the number of vacuolated nucleoli increased gradually from 24 % after 1.5 h up to 40 % after 24 h, while in the control there were 18 % of nucleoli with vacuoles and after 4-d chilling only 5 %. Labelling of cells during 20-min incubation in ³H-uridine and during 80-min post-incubation in non-radioactive medium was increased in recovered plants in comparison with the control and chilled plants. The conclusion has been drawn that nucleolar vacuoles in soybean plants are formed as a result of migration of granular component accumulated in nucleolus during 4-d chilling.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Dioscorea zingiberensis</Emphasis> Wright,an important pharmaceutical crop

Dioscorea zingiberensis Wright has been cultivated as a pharmaceutical crop for production of diosgenin, a precursor for synthesis of various important steroid drugs. Because breeding of D. zingiberensis through sexual hybridization is difficult due to its unstable sexuality and differences in timing of flowering in male and female plants, gene transfer approaches may play a vital role in its genetic improvement. In this study, the Agrobacterium tumefaciens-mediated transformation of D. zingiberensis was investigated with leaves and calli as explants. The results showed that both leaf segments and callus pieces were sensitive to 30 mg/l hygromycin and 50–60 mg/l kanamycin, and using calli as explants and addition of acetosyringone (AS) in cocultivation medium were crucial for successful transformation. We first immersed callus explants in A. tumefaciens cells for 30 min and then transferred the explants onto a co-cultivation medium supplemented with 200 μM AS for 3 days. Three days after, we cultured the infected explants on a selective medium containing 50 mg/l kanamycin and 100 mg/l timentin for formation of kanamycin-resistant calli. After the kanamycin-resistant calli were produced, we transferred them onto fresh selective medium for shoot induction. Finally, the kanamycin resistant shoots were rooted and the stable incorporation of the transgene into the genome of D. zingiberensis plants was confirmed by GUS histochemical assay, PCR and Southern blot analyses. The method reported here can be used to produce transgenic D. zingiberensis plants in 5 months and the transformation frequency is 24.8% based on the numbers of independent transgenic plants regenerated from initial infected callus explants.     

The Distribution of Garlic Viruses in Leaves and Bulbs during the First Year of Infection

Garlic plants are naturally infected with a mixture of viruses. Virus‐free garlic plants, obtained by meristem culture, rapidly become reinfected when planted in the field. With the aim of understanding virus movement and fluctuations in virus concentration in leaves and cloves of garlic plants in the first year after infection, Onion yellow dwarf virus, Leek yellow stripe virus, and other viruses were analyzed by double‐antibody sandwich enzyme‐linked immunosorbent assay. Significant differences were detected in virus concentration in different leaves, but the distribution of the viruses was variable. Therefore, no one type or position of leaf is preferable for detecting virus presence. Instead, sampling any leaf at the end of the crop cycle, about 200 days after planting, is advisable because virus concentration is several times higher in older plants. The analysis of virus distribution in bulbs revealed that virus concentration was higher in early‐inoculated than in late‐inoculated plants. In 81% of the bulbs, cloves were either all positive or all negative in serological tests. Only in 6% of the cases were positive and negative cloves found in the same bulb, and in 13% of the bulbs, negative results coexisted with an uncertain status. The tests of virus concentration in relation to the layers of each bulb revealed important differences. Only the innermost layer showed differences with other layers, but this was poorly represented as it had fewer cloves.     

Ectopically expressed leaf and bulb lectins from garlic (<Emphasis Type="Italic">Allium sativum</Emphasis> L.) protect transgenic tobacco plants against cotton leafworm (<Emphasis Type="Italic">Spodoptera littoralis</Emphasis>)

The insecticidal activity of the leaf (ASAL) and bulb (ASAII) agglutinins from Allium sativum L. (garlic) against the cotton leafworm, Spodoptera littoralis Boisd. (Lepidoptera: Noctuidae) was studied using transgenic tobacco plants expressing the lectins under the control of the constitutive CaMV35S promoter. PCR analysis confirmed that the garlic lectin genes were integrated into the plant genome. Western blots and semi-quantitative agglutination assays revealed lectin expression at various levels in the transgenic lines. Biochemical analyses indicated that the recombinant ASAL and ASAII are indistinguishable from the native garlic lectins. Insect bioassays using detached leaves from transgenic tobacco plants demonstrated that the ectopically expressed ASAL and ASAII significantly (P < 0.05) reduced the weight gain of 4th instar larvae of S. littoralis. Further on, the lectins retarded the development of the larvae and their metamorphosis, and were detrimental to the pupal stage resulting in weight reduction and lethal abnormalities. Total mortality was scored with ASAL compared to 60% mortality with ASAII. These findings suggest that garlic lectins are suitable candidate insect resistance proteins for the control of S. littoralis through a transgenic approach.     

In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree

Padar (Stereospermum personatum, family Bignoniaceae) is a well-known medicinal tree. Its complete regeneration occurred through shoot bud culture in vitro. The seeds germinated sequentially on plastic trays and polyethylene bags for 21 days served as explants source. Nodal segments from the seedlings were established on MS medium supplemented with 4.44 μM BA, in which 86.6% nodes showed shoot bud elongation. Then, nodal segments from the developed shoots were cultured on MS medium with several BA concentrations; best shoot multiplication was obtained with 0.44 μM BA. In a second experiment where PVP was added to proliferation medium, nodal segments from developed shoots produced maximum 2.78 shoots per node. The nodal segments showed shoot multiplication up to seventh subculture on. Finally, shoots were rooted on MS medium with 2.46 μM IBA. The plants transferred to net pots containing coco-peat were acclimatized in green house, where more than 80% plants survived and grew normally.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

The effect of asparagus virus infection on asparagus tissue culture

The impact of asparagus virus I (AV-I), a potyvirus, and asparagus virus II (AV-II), an ilarvirus, on micropropagation of field-grown asparagus was studied. Apical shoot tips excised from singly or doubly-infected plants were slow to develop roots and had a 15 to 75% reduction in survival in culture, respectively, compared to those excised from virus-free plants. The four virus infection groups were ranked: virus-free >AV-II>AV-I>AV-I & II for capacity of explants to both root and survivein vitro. Micropropagated plants infected with AV-II exhibited slight reductions in fresh and dry weights, with greater reductions associated with infection with AV-I and double infection, compared to the virus-free controls. Eighty-one virus-infected apical shoot tips yielded 7 (8.6%) virus-free clones, as determined by rub inoculation on indicator plants.     

Regeneration and transformation in adult plants of Campanula species

Adult plants are known for recalcitrance when it comes to adventitious organ formation and regeneration. Methods used for regeneration in explants from seedlings of Campanula carpatica failed to work for explants from adult plants of the same species. The present investigation generated efficient regeneration methods for mature specimens of four species of Campanula, C. carpatica, C. haylodgensis, C. portenschlagiana and C. poscharskyana. Petiole explants from dark-grown in vitro shoot cultures grown from nodal cuttings of adult plants regenerated successfully (95%), while explants from light-grown in vitro shoot cultures and greenhouse-grown plants regenerated at 12% and zero percentage, respectively. Dark-treatment, along with media manipulation with plant growth regulators, further enhanced regenerative capacity of the explants. A MS-based medium containing 10mg l ⁻¹ TDZ and 0.25 mg l⁻¹ NAA was the most efficient regeneration medium. Transgenic shoots from C. carpatica (3%) and C. haylodgensis (1%) and transgenic callus from all species were produced using Agrobacterium tumefaciens, and transformation was confirmed by histochemical and Southern blot analyses. Protocols developed in this study may be useful for achieving efficient regeneration and transformation of recalcitrant adult plants.