Embryonic Axis Explants Research Articles

Development of an Efficient and Reproducible Embryonic Axis-targeted Tissue Culture-based Transformation Protocol for Pigeonpea (Cajanus cajan)

Background: Tissue culture-based transformation approach poses significant challenges for genetic modification of recalcitrant pigeonpea due to poor regeneration efficiency. In this study, we present a comprehensive high-frequency regeneration and transformation protocol using embryonic axesas explants. Methods: Optimization of transformation parameters and culture conditions for different stages of adventitious shoot regeneration, including shoot induction, elongation and rooting, significantly enhanced transformation efficiency. Agrobacterium-mediated transformation was carried out using embryonic axes explants with pCAMBIA 2301 vector. Culturing of explants on MS basal media supplemented with BAP 2.5 in Preculture, BAP 2.5 in Selection Medium I, IAA 0.5, GA 0.5 and Zeatin 1 in Selection Medium II resulted in formation of numerous multiple shoots from primordia-forming calli at axillary bud regions. Result: Shooting efficiency was 82% and 77%, for cultivars, Pusa 992 and Asha respectively. Rooting efficiency was nearly 100% for both when cultured on half-strength MScontaining 15 g/l sucrose and 0.5 mg/l IAA. Regenerated plants exhibited a high level ofsurvival of 90% upon transplantation into pots. Successful gene integration and expression were confirmed through GUS histochemical and molecular analyses, with approximately 57% and 65% transformation efficiencies for cultivars Pusa 992 and Asha, respectively. This study presents a robust and reproducible tissue culture-based transformation system for pigeonpea, applicable across diverse genotypes, offering new possibilities for genetic improvement in this crucial legume crop.

Evaluation of transgenic chickpea harboring codon-modified Vip3Aa against gram pod borer (Helicoverpa armigera H.).

The gram pod borer is a major pest of chickpea, accounting for average annual yield losses to the tune of 40–50%. VIP3Aa, a class of insecticidal protein with different receptor binding site in the insect’s midgut compared to Bt-crystal protein, offers an alternative protection strategy against Lepidopteran insects. Here, we report evaluation of genetically engineered chickpea lines harboring codon modified Vip3Aa (cmVip3Aa) against the Lepidopteran insect pest, gram pod borer. The synthetic codon modified, cmVip3Aa gene of 2,370 bp was sub-cloned in modified plant expression vector and used for direct transformation of embryonic axis explants of chickpea (cv. DCP 92–3), with transformation efficiency of 4.30%. Presence and transmission of transgene across two generations were confirmed by PCR and Southern blot analyses in the five selected transgenic chickpea lines. Real Time PCR analyses indicated variable levels of cmVip3Aa expression in the transgenic chickpea lines (average Cq values 15.01±0.86 to 19.32±0.10), which were absent in the non-transgenic counterpart. Detached leaf insect bioassay indicate larval mortality (up to 39.75%), reduced larval feeding (up to 82.91%) and reduced larval weight gain (up to 68.23%), compared to control lines. Evaluation of gene offers a platform to identify efficacious insecticidal gene that can be used for insect resistance management in chickpea.

Genetic transformation of common beans (Phaseolus vulgaris L.) through Agrobacterium tumefaciens carrying Cry1Ab gene.

Seed beetles are one of the most important causes of yield loss in bean production. It is essential to develop resistant varieties in the fight against these pests. Agrobacterium-based gene transformation is the most widely used breeding method worldwide to develop insect-resistant varieties. Embryonic axes and plumule explants were obtained from Agrobacterium tumefciens treated mature zygotic embryos of low and high raw protein-based common bean cultivars Akman 98 and Karacaşehir 90. Agrobacterium tumefaciens contained a synthetic Bacillus thuringiensis insecticidal crystal protein gene (Bt Cry1Ab) controlled by the 35S promoter and NOS terminator sequences. The transformation event was genotype and explant dependent. The plumule explants could not withstand kanamycin-based selection pressure and died. It was possible to get two transgenic plants using embryonic axis explants of low protein cultivar Akman 98. These results were validated using GUS analysis, PCR, RT-PCR, bioassay analysis, and ELISA test from the samples taken from T0 and T1 generations. Bioassay tests showed that these plants were protected from the damage of legume seed insects (Bruchus spp.). The results are very encouraging and may help in producing better transgenic common bean germplasm leading to safe agriculture and reducing environmental pollutions.

Somatic embryogenesis and β-glucuronidase transformation in chickpea (Cicer arietinum cv. Bivanich).

Chickpea (Cicer arietinum L.) is an important kernel legume in the world. To optimize the plant tissue culture some experiments such as direct regeneration, proliferation, rooting shoots and somatic embryogenesis were done. In experiments were used direct regeneration and proliferation, various levels of plant growth regulators NAA (0 and 1.0mg/l), BAP (0, 1, 3 and 5mg/l) and three explants' types (epicotyl, cotyledon and embryonic axis). The results of both experiments showed that embryonic axis explant was better than other explants. The highest percentage was obtained in MS media containing 1mg/l BAP and also 3mg/l BAP and 0.1mg/l NAA with an average of 72%. The highest average number of branches (4.66) was found in the proliferation of embryonic axis in MS medium containing 3mg/l BAP. The highest rooting shoot (90%) was found in 1/2MS in B5 medium vitamins with 0.2mg/l of IBA and 0.5mg/l NAA. Somatic embryogenesis experiments were compared on the concentration gradient of 2,4-D in fine embryonic axis explants. The results displayed that the concentration gradient of 10mg/l 2,4-D to 5mg/l of 2,4-D and then to zero concentration showed the highest number of embryos. The best environment for regeneration embryos was MS medium with 2.5mg/l of 2,4-D concentration gradient to zero. In this study, the PCR reaction showed the presence of the β-glucuronidase (gus) marker gene in regenerated cotyledons for 20min in all three strains studied.

Multiple Shoot Regeneration from Detached Embryonic Axis in Greengram (Vigna radiata) cv.SML 668

Background: Under prevailing regime of climate change and dynamic pest-crop interactions, greengram genetic improvement requires a speedier and precise tool such as genetic transformation to develop improved lines. In this context, an in vitro multiple shoot regeneration system via organogenesis was demonstrated in mungbean cv. SML668, a parent cultivar used in crop improvement programs, using double cotyledonary node (DCN) and detached embryonic axis (EA) explants with 1.0 mg l-1 BAP. Methods: While both the explants responded to in vitro regeneration, number of shoots regenerated was higher with EA (4.02±0.19) than with DCN (3.1±0.08). 6-benzyl aminopurine (BAP) was found to be most effective for inducing and regenerating multiple shoots, in comparison to all other phytohormones (NAA, IAA and TDZ) and supplements (amino acids) tested. Sub culturing twice on BAP supplemented media followed by two subcultures on basal media was optimal for multiple shoot regeneration. Rhizogenesis was obtained on basal media devoid of any phytohormones in EA explants and in 1.0 mg l-1 IAA for DCN explants. The in vitro regenerated plantlets were successfully hardened in a mixture of soil, sand and vermiculite that flowered, produced pods and viable seeds on maturity. Result: The study revealed that detached embryonic axis was a potential explant for in vitro regeneration in SML668, a cultivar not tested for its in vitro regeneration ability before.

Developing a rapid and highly efficient cowpea regeneration, transformation and genome editing system using embryonic axis explants.

SUMMARYCowpea (Vigna unguiculata (L.) Walp.) is one of the most important legume crops planted worldwide, but despite decades of effort, cowpea transformation is still challenging due to inefficient Agrobacterium‐mediated transfer DNA delivery, transgenic selection and in vitro shoot regeneration. Here, we report a highly efficient transformation system using embryonic axis explants isolated from imbibed mature seeds. We found that removal of the shoot apical meristem from the explants stimulated direct multiple shoot organogenesis from the cotyledonary node tissue. The application of a previously reported ternary transformation vector system provided efficient Agrobacterium‐mediated gene delivery, while the utilization of spcN as selectable marker enabled more robust transgenic selection, plant recovery and transgenic plant generation without escapes and chimera formation. Transgenic cowpea plantlets developed exclusively from the cotyledonary nodes at frequencies of 4% to 37% across a wide range of cowpea genotypes. CRISPR/Cas‐mediated gene editing was successfully demonstrated. The transformation principles established here could also be applied to other legumes to increase transformation efficiencies.

An Efficient Agrobacterium-Mediated Genetic Transformation Using Embryonic Axis in Cotton (Gossypium hirsutum L.)

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

Improved salinity tolerance and growth performance in transgenic sunflower plants via ectopic expression of a wheat antiporter gene (TaNHX2).

Sunflower (Helianthus annuus. L) is one of the principal oil seed crops affected by the salinity stress, which limits the oil content and crop yield of sunflower plants. The acclimatization of plants to abiotic stresses such as salinity tolerance is mainly mediated by the vacuolar Na+/H+ antiporters (NHX) by tagging Na+ into vacuoles from the cytosol. We show here that the over-expression of wheat TaNHX2 gene in transgenic sunflower conferred improved salinity stress tolerance and growth performance. Transgenic sunflower plants were produced by infecting the embryonic axis ex-plants with Agrobacterium tumefaciens strain EHA105 containing a pBin438-TaNHX2 binary vector that carried a wheat antiporter (TaNHX2) gene under the control of a double CaMV 35S promoter with NPT II gene as a selectable marker. PCR analysis of T0 and T1 transgenic plants confirmed the integration of TaNHX2 in sunflower genome. Stable integration and expression of TaNHX2 in sunflower genome was further verified by Southern hybridization and semi-quantitative RT-PCR analyses. As compared to the non-transformed plants, TaNHX2 expressing transgenic plants showed better growth performance and accumulated higher Na+, K+ contents in leaves and roots under salt stress (200mM NaCl). Transgenic sunflower plants displayed improved protection against cell damage exhibiting stable relative water content, chlorophyll content, increased proline accumulation and improved reactive oxygen species (ROS) scavenging because of higher activities of the antioxidant enzymes like superoxide dismutase and ascorbate peroxidase, along with decreased production of hydrogen peroxide, free oxygen radical and malondialdehyde (MDA) under salt stress (200mM NaCl). Taken together, our findings suggest that TaNHX2 expression in sunflower plants contributed towards improving growth performance under sodium chloride stress.

Transgenic pigeonpea events expressing Cry1Ac and Cry2Aa exhibit resistance to Helicoverpa armigera.

Independent transgenic pigeonpea events were developed using two cry genes. Transgenic Cry2Aa-pigeonpea was established for the first time. Selected transgenic events demonstrated 100% mortality of Helicoverpa armigera in successive generations. Lepidopteran insect Helicoverpa armigera is the major yield constraint of food legume pigeonpea. The present study was aimed to develop H. armigera-resistant transgenic pigeonpea, selected on the basis of transgene expression and phenotyping. Agrobacterium tumefaciens-mediated transformation of embryonic axis explants of pigeonpea cv UPAS 120 was performed using two separate binary vectors carrying synthetic Bacillus thuringiensis insecticidal crystal protein genes, cry1Ac and cry2Aa. T0 transformants were selected on the basis of PCR and protein expression profile. T1 events were exclusively selected on the basis of expression and monogenic character for cry, validated through Western and Southern blot analyses, respectively. Independently transformed 12 Cry1Ac and 11 Cry2Aa single-copy events were developed. The level of Cry-protein expression in T1 transgenic events was 0.140-0.175% of total soluble protein. Expressed Cry1Ac and Cry2Aa proteins in transgenic pigeonpea exhibited significant weight loss of second-fourth instar larvae of H. armigera and ultimately 80-100% mortality in detached leaf bioassay. Selected Cry-transgenic pigeonpea events, established at T2 generation, inherited insect-resistant phenotype. Immunohistofluorescence localization in T3 plants demonstrated constitutive accumulation of Cry1Ac and Cry2Aa in leaf tissues of respective transgenic events. This study is the first report of transgenic pigeonpea development, where stable integration, effective expression and biological activity of two Cry proteins were demonstrated in subsequent three generations (T0, T1, and T2). These studies will contribute to biotechnological breeding programmes of pigeonpea for its genetic improvement.

Regeneration of plantlets from immature explantsculture in [Glycine max (L.) Merrill

For plant regeneration through somatic embryogenesis, immature cotyledonary nodes and embryonic axes explants of soybean cultivars JS 335 were cultured on six different medium fortified with MS salts, B5 vitamins and combinations of growth regulators. Between explants, immature embryonic axes demonstrated better regeneration potential as compared to cotyledonary node. For immature embryonic axes culture, better morphogenic callus induction, embryogenesis, organogenesis and plantlet regeneration was observed on MS with 4 B1.86N, MS 10N, MS .1B1N and MS.2B1N as compared to other media formulations. Cotyledonary nodes exhibited higher callus induction, embryogenesis, organogenesis on media with MS4 B1.86N, MS .1B1N, MS 3B6N and higher plantlet regeneration on media with MS.5B1N.

Effects of pulsed magnetic field treatment of soybean seeds on calli growth, cell damage, and biochemical changes under salt stress

The effects of magnetic field (MF) treatments of soybean seeds on calli growth, cell damage, and biochemical changes under salt stress were investigated under controlled conditions. Soybean seeds were exposed to a 1.0 Hz sinusoidal uniform pulsed magnetic field (PMF) of 1.5 µT for 5 h/day for 20 days. Non-treated seeds were considered as controls. For callus regeneration, the embryonic axis explants were taken from seeds and inoculated in a saline medium with a concentration of 10 mM NaCl for calli growth analysis and biochemical changes. The combined treatment of MF and salt stress was found to significantly increase calli fresh weight, total soluble sugar, total protein, and total phenol contents, but it decreased the ascorbic acid, lipid peroxidation, and catalase activity of calli from magnetically exposed seeds compared to the control calli. PMF treatment significantly improved calli tolerance to salt stress in terms of an increase in flavonoid, flavone, flavonole, alkaloid, saponin, total polyphenol, genistein, and daidzein contents under salt stress. The results suggest that PMF treatment of soybean seeds has the potential to counteract the adverse effects of salt stress on calli growth by improving primary and secondary metabolites under salt stress conditions.

Agrobacterium-mediated genetic transformation of pigeon pea [Cajanus cajan (L.) Millsp.] for resistance to legume pod borer Helicoverpa armigera

Agrobacterium-mediated genetic transformation was performed using embryonic axes explants of pigeon pea. Both legume pod borer resistant gene (cry1Ac) and plant selectable marker neomycine phosphor transferase (nptII) genes under the constitutive expression of the cauliflower mosaic virus 35S promoter (CaMV35S) assembled in pPZP211 binary vector were used for the experiments. An optimum average of 44.61% successfully hardened dot blot Southern hybridization positive plants were obtained on co-cultivation media supplemented with 200 μM acetosyringone without L-cysteine. The increased transformation efficiency from a baseline of 11.53% without acetosyringone to 44.61% with acetosyringone was further declined with the addition of different concentrations of L-cysteine to co-cultivation media. Transgenic shoots were selected on 50 and 75 mg L−1 kanamycin. Rooting efficiency was 100% on half-strength Murashige and Skoog medium with 20 g L−1 sucrose and 0.5 mg L−1 indole butyric acid in the absence of kanamycin. Furthermore, 100% seed setting was found among all the transgenic events. The plants obtained were subjected to multi- and nochoice tests to determine the behavioral responses and mortality through Helicoverpa armigera bioassays on the leaf and relate their relationship with the expression of cry1Ac protein which was found to be less in leaf as compared to the floral buds, anther, pod, and seed.

In vitro regeneration through organogenesis and somatic embryogenesis in pigeon pea [ Cajanus cajan (L.) Millsp.] cv. JKR105.

In vitro regeneration of pigeon pea through organogenesis and somatic embryogenesis was demonstrated with pigeon pea cv. JKR105. Embryonic axes explants of pigeon pea showed greater regeneration of shoot buds on 2.5mgL(-1) 6-benzylaminopurine (BAP) in the medium, followed by further elongation at lower concentrations. Rooting of shoots was observed on half-strength Murashige and Skoog (MS) medium with 2% sucrose and 0.5mgL(-1) 3-indolebutyric acid (IBA). On the other hand, the regeneration of globular embryos from cotyledon explant was faster and greater with thidiazuron (TDZ) than BAP with sucrose as carbohydrate source. These globular embryos were maturated on MS medium with abscisic acid (ABA) and finally germinated on half-strength MS medium at lower concentrations of BAP. Comparison of regeneration pathways in pigeon pea cv. JKR105 showed that the turnover of successful establishment of plants achieved through organogenesis was more compared to somatic embryogenesis, despite the production of more embryos than shoot buds.

In vitro plant regeneration from decapitated embryonic axes of Clitoria ternatea L.—An important medicinal plant

In vitro clonal propagation of Clitoria ternatea has been achieved by employing decapitated embryonic axes (DEAs) explants. The explants induced multiple shoots on cytokinin-containing medium. Several cytokinins [6-benzylaminopurine (BAP), 6-furfuryl aminopurine (KIN) and thidiazuron (TDZ)] were assayed. The best response was achieved with 2 mg l −1 BAP in which 100% of cultures produced 6.0 ± 0.14 shoots per explant. MS + 1 mg l −1 gibberellic acid (GA 3) was the most suitable for shoot elongation. Regenerated shoots were rooted in half-strength Murashige and Skoog (MS) medium with 0.2 mg l −1 indole-3-butyric acid (IBA). Plantlets were successfully acclimatized and established in soil, and they were morphologically indistinguishable from the source plant. The plantlets attained maturity and flowered normally. The efficient regeneration protocol reported here provides an important method of micropropagation of this plant. Furthermore, this protocol may be used for genetic transformation of this valuable medicinal plant for its further improvement.

In vitro shoot regeneration from preconditioned explants of chickpea (Cicer arietinum L. ) cv. Gokce

The present study reports the successful shoot regeneration of preconditioned mature embryo and embryonic axis explants of chickpea cv. Gokce . Explants were preconditioned with 10 mgl benzylaminopurine (BA) for 7 days followed by culture on Murashige and Skoog (MS) medium containing 0.25, 0.50, 1.00 and 2.00 mg/l BA with or without 0.25 mg/l naphthalene acetic acid (NAA) supplemented with 4 mg/l activated charcoal and 1 mg/l polyvinylpyrrolidon (PVP). Shoot regeneration was recorded on all explants. Maximum number of (14.75) shoots per explants on mature embryo were recorded on MS medium containing 2.0 mg/l BA with 0.25 mg/l NAA. Whereas, 16.83 shoots per explants were recorded on MS medium containing 2.0 mg/l BA. Presence of NAA in the culture medium decreased the mean shoot length of both explants compared to medium devoid of 0.25 mg/l NAA. Regenerated shoots were rooted on MS medium containing 1.0 mg/l indole-butyric acid (IBA) after 4 weeks of culture. Rooted plantlets were transferred to pots for acclimatization under green house conditions. Key words : In vitro, chickpea, pulse treatment, shoot regeneration.

Regeneration efficiency of cowpea (Vigna unguiculata (L.) Walp.) via embryonic axes explants

The experiment was conducted to test the in vitro regeneration efficiency of cowpea shoots via embryonic axes explants and the effect of benzyl amino purine (BAP) concentration on shoot formation. A total of 600 explants (embryonic axes) were plated from which 237 shoots were regenerated. The average regeneration ratio was 39% and the number of shoot per explant obtained stood at an average of 6.0. This indicates likelihood that higher regeneration percent and number of shoots per explant could be possible as more proficiency is attained in the use of the protocol. The role of the growth regulator, benzyl amino purine (BAP), as a shoot growth inducing factor and modifier in cowpea in vitro regeneration was demonstrated in the experiment. The protocol used in the experiment gave promising results for the efficient regeneration and subsequent genetic transformation of cowpea. Key words: Cowpea, in vitro, regeneration ratio, benzyl amino purine (BAP), embryonic axes explants.

Efficient Embryogenic Callus Induction and Plant Regeneration from Embryonic Axis Explants in Quercus acutissima

Embryogenic callus of Quercus acutissima was successfully induced from embryogenic cultures, and plants were regenerated from the callus. The development of the techniques involved will allow mass propagation and gene transformation in this species. Embryogenic cultures were formed from embryonic axis explants (i.e., embryos without cotyledons) excised from immature embryos, after culture on Murashige and Skoog (MS) medium containing indolebutyric acid and benzyladenine. Attempts to induce embryogenic cultures from cotyledon explants were unsuccessful. Embryogenic calli were induced at high frequency from embryogenic cultures on MS medium containing 2,4-dichlorophenoxyacetic acid. However, benzyladenine inhibited embryogenic callus formation. Somatic embryo development from embryogenic calli occurred on MS medium in all of the seven cell lines tested. Germination of somatic embryos was induced on half strength MS medium without plant growth regulators. Finally, acclimated plants growing in soil were obtained.

Transgenic yellow lupin (Lupinus luteus).

Transgenic yellow lupin (Lupinus luteus L.) plants have been generated by meristem co-cultivation with Agrobacterium tumefaciens. The binary plasmid pPZBNIa contains the bar gene under the control of a CaMV 35 S promoter. The transformation method involves inoculation of embryonic axis explants with A. tumefaciens, flooding the meristem with glufosinate, and initial culture on non-selective medium. Shoots were transferred to culture medium containing 20 mg/l glufosinate. Following subculture, shoots were grafted onto non-transgenic narrow-leafed lupin (L. angustifolius L.) seedling rootstocks, or rooted in vitro. The overall transformation efficiency, as determined at the T1 generation, was 0.05%-0.75%. The transgenic nature of plants grown to the T6 generation was confirmed by phosphinothricin acetyl transferase, PCR and Southern analyses.

Plant regeneration from embryo-derived callus in Phaseolus vulgaris L. (common bean) and P. acutifolius A. Gray (tepary bean).

Regeneration-competent callus of Phaseolus vulgaris and P. acutifolius was obtained from mature embryo explants on a medium containing thidiazuron and indole-3-acetic acid. For the P. vulgaris genotype Xan-159, regeneration was achieved from cotyledon explants, but not from embryonic axis explants. Both explants could be used for the P. acutifolius genotype NI 574 but embryonic axes gave the best results. In-vitro-rooted plantlets of P. acutifolius could readily be established in the greenhouse. For P. vulgaris hardening problems with in-vitro-rooted plantlets could be overcome by means of in vitro grafting. The potential of the described procedure for obtaining transgenic P. vulgaris plants is discussed.

An Efficient in vitro Regeneration System for Australian-Grown Chickpea (Cicer arietinum) Cultivars

A comparison of methods for efficient in vitro regeneration of Australian-grown chickpea (Cicer arietinum L.) cultivars was undertaken. The most efficient regeneration system was one where immature cotyledon and embryonic axis explants, 14-21 days post-pollination, were cultivated on Murashige and Skoog's salts with Gamborg's vitamins, 1.0, 3.0 or 5.2 mg L-1 zeatin, 0 or 35 μg L-1 indole-3-acetic acid, 30 g L-1 sucrose and 8 g L-1 Phytagar. The first embryoid structures appeared after 2 weeks of culture at 25 ± 1°C in dim light (150 μmol m-2 s-1) and formed directly on the edges of the immature cotyledons or petiole stumps. Between 10 and 20 structures were produced on each cotyledon explant in two cultivars, however, the embryogenic structures which developed on cv. Narayen were more efficiently transformed into shoots than far cv. Amethyst. An efficient regeneration medium (2 mg L-1 naphthaleneacetic acid, 1/2 Murashige and Skoog's salts with Gamborg's vitamins, and 0.5 g L-1 activated charcoal) was used to develop a portion of the shoots into morphologically normal plants growing in a vermiculite and soil potting mix in a growth room. Less efficient in vitro regeneration was observed when hypocotyl and shoot sections, and shoot apices were induced to form callus and plants by organogenesis. These plants could not be established in a potting mix. The amount and type of callus produced varied between explant type and cultivar.