Intermediate Callus Phase Research Articles

Cryopreservation of aromatic ginger Kaempferia galanga L. by encapsulation-dehydration

Kaempferia galanga L. is an endangered multi-purpose medicinal plant in Family Zingiberaceae, the rhizomes of which are used for several ayurvedic formulations. Encapsulation-dehydration (ED) method was optimized for cryopreservation of shoot tips of K. galanga. Shoot tips (STs) bearing the apical meristem dissected from the established in vitro shoot cultures were preconditioned in MS+0.4 M sucrose prior to encapsulation in calcium alginate and the beads subsequently transferred to MS liquid+0.3 M sucrose for 3 days afterward dehydration inside the laminar airflow for 4 hours upon rapid freezing in LN and rapid thawing produced maximum 62.2% survival and 46.7% regeneration rates. Shoot regeneration was observed from the apical meristems exclusive of intermediary callus phase. The plantlets regenerated from cryopreserved STs transferred to the field were phenotypically analogous with the mother plant.

Morphological and Genetic Stability of Dormant Apple Winter Buds After Cryopreservation

Twenty apple germplasm accessions from the Korean Genebank were successfully cryopreserved using two-step freezing to back up genetic resources maintained by field collections. This study examined the morphological and genetic stability of cryopreserved dormant apple buds that were stored in liquid nitrogen, and then rewarmed and regrown. Whole plants were regenerated directly from dormant buds through budding without an intermediary callus phase. The cryopreserved buds produced high levels of shoot formation (76.2-100%), similar to those of noncryopreserved buds (91.3-100%), with no observed differences between cryopreserved and noncryopreserved materials. Three of the twenty cryopreserved apple germplasm accessions were used to assess morphological and genetic stability. No differences in morphological characteristics including shoot length, leaf shape, leaf width/length ratio, and root length were observed between controls (fresh control and noncryopreserved) and cryopreserved plantlets. The genetic stability of regenerants (before and after cryopreservation) was investigated using inter simple sequence repeat (ISSR) markers. The ISSR markers produced 253 bands using four primers, ISSR 810, SSR 835, ISSR 864, and ISSR 899. These markers showed monomorphic banding patterns and revealed no polymorphism between the mother plant and regenerants before and after cryopreservation, suggesting that cryopreservation using two-step freezing does not affect the genetic stability of apple germplasm. These results show that two-step freezing cryopreservation is a practical method for long-term storage of apple germplasms.

Plantlet formation via somatic embryogenesis and LC ESI Q-TOF MS determination of secondary metabolites in Butea monosperma (Lam.) Kuntze

Medicinal properties of Butea monosperma (BM) and overexploitation of bark as a rich source of flavonoids for different biological activities, development of efficient method for high frequency somatic embryos and in vitro synthesis of bioactive secondary metabolites using plant tissue culture technology is important. Initially, callus was induced from leaf explants of BM on Murashige and Skoog (MS) medium containing 0.25 mg L−1 2,4-d-dichlorophenoxyacetic acid (2,4-d) with 0.1 mg L−1 kinetin (Kn) and ascorbic acid (AA). MS half strength macronutrients and full strength micronutrients containing 0.25 mg L−1 2,4-d with 0.1 mg L−1 Kn, and 0.5 mg L−1 AA provided fragile callus with 84.0 ± 1.00 % optimal growth response. Shoot formation occurred via somatic embryogenesis through an intermediary callus phase. However, 2.1 mg L−1 thidiazuron with 0.5 mg L−1 AA provides high frequency (79.6 ± 2.02 %) of somatic embryogenesis within 5 weeks. Developed embryos when transferred to woody plant medium containing 0.5 mg L−1 AA with 3.0 mg L−1 Kn and 0.5 mg L−1 α naphthalene acetic acid responded 44.0 ± 0.00 % embryo maturation, whereas 0.5 mg L−1 Kn, 0.3 mg L−1 indole-3-butyric acid, and 0.25 mg L−1 AA induced rooting within 6 and 8 weeks, respectively. Liquid chromatography electro spray ionization quadrupole time of flight mass spectrometry (LC ESI Q-TOF MS) analysis of in vitro cultures showed similarity to those compounds identified in wild grown leaf samples known for osteogenic activity. Histological investigation through scanning electron microscopy demonstrates the developmental stages of somatic embryos, shoot bud formation, and induction of root primordial.

High frequency in vitro plantlet regeneration and antioxidant activity of Enicostema axillare(Lam.) Raynal ssp. littoralis (Blume) Raynal: An important medicinal plant

Abstract Objective To develop a method for high frequency plantlets regeneration protocol for Enicostema axillare (Lam.) Raynal ssp. littoralis (Blume) Raynal ( E. axillare ) without intermediate callus phase and to determine the content and correlation of phenolic compounds and their antioxidant activity of both the plants derived from nature and nodal culture by DPPH assay. Methods The nodal explants were cultured on MS basal medium fortified with different concentration of various growth regulators such as BAP, KIN, TDZ and 2iP (0.5–2 mg/L) individually and in combinations with or without GA 3 for shoot bud induction and multiplication. Total phenol and flavonoid content was determined in both plants from nature and nodal culture and antioxidant activity was determined by DPPH assay. Results The highest number of multiple shoot (108.00±3.55 shoots/explants) was obtained on MS medium supplemented with BAP (2 mg/L) in combination with KIN (0.5 mg/L) and GA 3 (2 mg/L). Rooting was optimized on half-strength MS medium supplemented with IAA (0.5 mg/L). The rooted plantlets were transferred to paper cups containing vermiculite and hardening was successfully attained with 75% survival. Among the four extract of methanol and water extract from both the plantlet from nature and nodal culture, the concentration of flavonoid was found to be higher in methanol extract of the plants from nature (146.57±1.68 mg rutin/g extract) and phenol content was higher in the water extract of plant from nature (52.53±1.67 mg GAE/g extract). The radical scavenging activity of four extracts. Methanol extract of plant grown in nature showed the highest radical scavenging activity (IC 50 = 87.10 μg/mL) was investigated by DPPH test. Conclusion The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps for high scale production of plantlets. A high correlation between antioxidant capacities and their total phenolic contents indicated that flavonoid compounds were a major contributor of antioxidant activity of both plants grown from nature and nodal culture.

Plant regeneration through direct somatic embryogenesis from immature zygotic embryos of the medicinal plant, Paris polyphylla Sm.

A protocol for induction of direct somatic embryogenesis and subsequent plant regeneration for the medicinally important and endangered plant Paris polyphylla Sm. has been developed for the first time. Immature zygotic embryos (IZEs) were cultured on different media namely Gamborg (B5), ½ B5, Murashige and Skoog (MS), ½ MS, Chu et al. (N6), ½ N6, Schenk and Hildebrandt (SH) and ½ SH. Highest frequency of somatic embryogenesis (32.6 %) and mean number of somatic embryos (SEs) per explant (28.7 ± 1.7) were obtained on ½ MS medium directly without an intermediate callus phase. The frequency of SE induction was significantly increased to 40.7 % when ½ MS medium was solidified with gelrite compared to agar (32.6 %). Secondary somatic embryos (SSEs) appeared on the primary SEs in a repetitive way on plant growth regulator-free ½ MS medium but with a gradual decrease in embryogenic potential during subsequent subcultures. Plasmolyzing pre-treatment of SSEs with 1.0 M mannitol for 12 h effectively maintains its embryogenic capacity. Primary embryos at the elongated dimpled and early cotyledonary stage displayed the highest embryo forming capacity of 26.94 and 27.87, respectively. High frequency of SE germination (94.0 %) occurred on ½ MS medium with 0.5 mg/l gibberellic acid. Highest percentage of seedling to plantlet conversion was observed in the medium supplemented with 0.05 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthalene acetic acid. Regenerated plants displayed morphological characteristics similar to that of the wild plants. Flow cytometry analysis showed ploidy stability of the regenerated plants.

English

Genetic stability of Morus alba, Morus indica, Morus laevigata (indigenous collection) and Morus species (exotic collection) have been studied in in vitro regenerated plants of mulberry (fresh, before and after cryopreservation) using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) markers. This study examined the genetic stability of cryopreserved dormant buds of Morusgermplasm that were stored in liquid nitrogen using two-step freezing, then rewarmed and regrown. Dormant buds of mulberry collected during winter period were found suitable for the cryopreservation in liquid nitrogen. In the present study, the plants were regenerated directly from dormant buds (before and after cryopreservation) without intermediary callus phase. These regenerants thus bear low risk of genetic instability. Both the single primer amplification reaction (SPAR) markers showed reproducible and well resolved banding patterns in mulberry germplasm, in which RAPD marker generated a total of 201 bands based on 15 primers; however, ISSR markers were given 145 bands using 11 primers. Both markers showed monomorphic banding patterns and did not reveal any polymorphism among the mother plant and in vitro regenerants before and after cryopreservation, suggesting that cryopreservation, using two-step freezing, does not affect genetic stability of mulberry germplasm. The transitory nature of these polymorphisms should be carefully considered when monitoring for genetic stability. Key words: Cryopreservation, Genetic stability, in vitro culture, ISSR, mulberry, RAPD.

High frequency microcloning of Aloe vera and their true-to-type conformity by molecular cytogenetic assessment of two years old field growing regenerated plants.

BackgroundAloe vera (L.) Burm.f is an important industrial crop, which has enormous application in pharmaceutical, cosmetic and food industries. Thereby, the demand for quality planting material of A. vera is increasing worldwide. Micropropagation is the widely accepted practical application of plant biotechnology that has gained the status of a multibillion-dollar industry throughout the world and this techniques can be used to meet the industrial demand of A. vera. Present studies aim to develop a proficient methods of high-frequency true-to-type plantlet regeneration without intermediate callus phase for A. vera.ResultsNodal portion of rhizomatous stem of A. vera were cultured on Murashige and Skoog (MS) medium (Physiol. Plant. 15:473 – 497, 1962) supplemented with various cytokinin and A. vera leaf gel (AvG) as organic supplement. Number of proliferated shoots per explant was increased along with the regeneration cycles and on MS medium supplemented with 2.5 mg/L 6-benzylaminopurine and 10.0% (v/v) AvG, only 17.8 ± 0.35 shoots per explant were induced on 1st regeneration cycle whereas on 3rd regeneration cycle these number increase to 38.5 ± 0.44 shoots per explant on the same medium composition. AvG have an encouraging role to increase the proliferation rate and on 3rd regeneration cycle 27.6 ± 0.53 shoot per explant induced on 2.5 mg/L BAP, but these number increase to 38.5 ± 0.44 shoots per explant when 10.0% (v/v) AvG was added along with 2.5 mg/L BAP. After transfer of individual excised shoots to a one-third strength MS medium containing 20.0% (v/v) AvG, all the shoots formed whole plantlets with maximum number (9.6 ± 0.29) of roots per shoot. 95.0% of the regenerated plantlets survived on poly-green house. Normal flower appeared in 84.2% field growing micropropagated plants after 18 to 20 months of field transfer. Further, clonal fidelity of the two years old micropropagated plants was established by studying mitotic and meiotic chromosomal behavior and also considered the chromosome number and structural organization. There were no alterations in chromosome phenotypes, somatic haploid (pollen mitosis) and diploid chromosome count (n = 7; 2n = 14), or meiotic behavior. Randomly amplified polymorphic DNA analyses revealed there were no somaclonal variations among these regenerants.ConclusionsThese results confirm the very reliable method for large scale production of true-to-type plantlets of A. vera, which can be used for commercial purpose.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-54-46) contains supplementary material, which is available to authorized users.

Proteomic Analysis of Callus Development in Vanilla planifolia Andrews

The orchid Vanilla planifolia has long been cultivated for its flavoured pods. Plant regeneration via an intermediary callus phase remains difficult, partially attributed to a low frequency of callus formation from various explants. In this study, a two-dimensional gel electrophoresis (2-DE) gel-based proteomic approach was taken to investigate the molecular mechanisms involved in callus formation and development. Protein extracts from 15-day-old callus with a mean weight of 20–30 mg (CS-1) and 45-day-old callus with a mean weight of 50–60 mg (CS-2) induced from nodal explants (NC) of V. planifolia were extracted and analysed using 2-DE. Protein spots detected on 2-D gels from NC, CS-1 and CS-2 were 265, 179 and 223 protein spots, respectively, with the majority being distributed across the isoelectric focusing point (pI) range of 5.0–6.9 and with molecular masses between 25–100 kDa. Of these, 73 protein spots showed significantly differential expression, and 23 proteins were successfully identified. The majority of proteins differing between nodal explants and callus tissues were classified as defence and stress response, metabolism, protein synthesis, transport, transcription, iron storage, photosynthesis and organ-specific proteins. The largest group of proteins identified were stress response proteins, indicating their effects on callus formation.

Propagation of goldenrod (Solidago canadensis L.) from leaf and nodal explants

Goldenrod (<em>Solidago canadensis </em>L.) is an invasive plant species in many countries except North America but a cut-flower species worldwide. There is a need to generate and propagate goldenrod clones efficiently for research and commercial purposes. A callus induction and plantlet regeneration system was developed by studying the influence of explant type and different concentrations of plant growth regulators. The highest callus production from leaf segments was obtained on Murashige and Skoog’s medium (MS medium) supplemented with 1.0 mg/L naphthalene acetic acid (NAA) and 1.0 mg/L 6-benzylaminopurine (BA). Adventitious shoots could be regenerated directly from leaf explants without an intermediate callus phase with the highest shoot induction percentage of 87.2%. The largest number of adventitious shoots per leaf explant (3.2) was obtained on MS medium supplemented with 0.4 mg/L NAA and 2.0 mg/L BA. MS medium supplemented with 0.1 mg/L NAA and 1.0 mg/L BA was the best medium for axillary shoot regeneration from nodal segments. The highest root number and longest roots occurred on half-strength MS without the addition of any growth regulator. Rooted plantlets were then transferred to a soil-based growth medium, placed in a greenhouse, and acclimatized with 100% success. All surviving plants grew normally without showing any morphological varia­tion when compared to those grow from seed. This regeneration protocol may be used to produce certain biotypes of goldenrod suitable for genetic transformation rapid propagation of goldenrod for commercial purposes or for screening fungi and toxins as potential biocontrol agents against this weed.

Assessment of Genetic Stability and Instability of Tissue Culture-Propagated Plantlets of Aloe vera L. by RAPD and ISSR Markers

Efficient plantlet regeneration with and without intermediate callus phase was achieved for a selected genotype of Aloe vera L. which is sweet in test and used as a vegetable and source of food. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) marker assays were employed to evaluate genetic stability of plantlets and validate the most reliable method for true-to-type propagation of sweet aloe, among two regeneration systems developed so far. Despite phenotypic similarities in plantlets produced through both regeneration systems, the differences in genomic constituents of plantlets produced through intermediate callus phase using soft base of inflorescence have been effectively distinguished by RAPD and ISSR markers. No polymorphism was observed in regenerants produced following direct regeneration of axillary buds, whereas 80% and 73.3% of polymorphism were observed in RAPD and ISSR, respectively, in the regenerants produced indirectly from base of the inflorescence axis via an intermediate callus phase. Overall, 86.6% of variations were observed in the plantlets produced via an intermediate callus phase. The occurrence of genetic polymorphism is associated with choice of explants and method used for plantlet regeneration. This confirms that clonal propagation of sweet aloe using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. On the other hand, a high degree of variations were observed in plantlets obtained through indirect regeneration and thus cannot be used for the mass multiplication of the genotype; however, it can be used for crop improvement through induction of somaclonal variations and genetic manipulations.

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.

Developing a scale-up system for the in vitro multiplication of thidiazuron-induced strawberry shoots using a bioreactor

The use of large-scale liquid cultures in a bioreactor system has the potential to resolve the manual handling of the various stages of micropropagation and increases shoot multiplication in vitro significantly compared with those cultured on semi-solid gelled medium. In an attempt to improve the micropropagation protocol for strawberry (Fragaria × ananassa Duch.), a procedure for the mass propagation of adventitious shoots regenerated from leaf, sepal and petiole explants of cultivar Bounty using a liquid medium-containing bioreactor system combined with gelled medium is described. Leaf disks, sepals and petiole halves produced multiple buds and shoots without an intermediary callus phase on 2-4 µM thidiazuron (TDZ)-containing shoot induction medium within 5-6 wk of culture initiation. TDZ supported rapid shoot proliferation at low concentrations (0.1 µM), but induced hyperhydricity in a bioreactor system. Bioreactor-multiplied hyperhydric shoots were transferred to gelled medium containing 2-4 µM zeatin, and produced normal shoots and root within 4 wk of culture. In vitro derived plantlets were acclimatized and eventually established in the greenhouse and in the field. Present results suggested the possibility of large-scale multiplication of strawberry shoots in bioreactors. Key words: Fragaria × ananassa, growth regulator, shoot regeneration, RITA® bioreactor

Combined Direct Regeneration Protocols in Tissue Culture of Different Cumin Genotypes Based on Pre-existing Meristems

Rapid and genotype-independent protocols for two direct in vitro morphogenesis pathways including direct shoot organogenesis from embryo and direct shoot proliferation from node have been developed in cumin (Cuminum cyminum L.). Direct regenerations occurring without passing callus phase are important since fewer somaclonal variation and genotype-dependency are likely to arise from these methods in comparison with regenerations trough callus. After embryo culture, shoots with single-cellular origin were regenerated from the meristematic zone of embryo without any intermediate callus phase. In contrast, proliferated shoots with multi-cellular origin were directly regenerated from the axillary buds (meristems) of node explants. Effects of different concentrations of 6-Benzylaminopurine (BAP), alpha-Naphthaleneacetic Acid (NAA) and Indole-3-kcetic Acid (IAA) on B5 medium of embryo and node cultures as well as subculture were studied in detail. In direct organogenesis pathway from embryo explant, 0.1 mg L(-1) NAA + 1 mg L(-1) IAA resulted the highest shoot regeneration response (89.5 shoots per regenerated explant), whereas 0.1 mg L(-1) BAP + 1 mg L(-1) NAA was the most effective combination in direct shoot proliferation from node explant (42 shoots per regenerated explant). BAP (cytokinin) revealed the inhibitory effect on induction of direct shoot organogenesis pathway from embryo explant, while low concentration of BAP (0.1 mg L(-1)) had positive effect on direct shoot proliferation pathway from node explant. Subculturing was not necessary for shoot multiplication and elongation in embryo culture, whereas multiplication and elongation of shoots in node culture were associated to subculture on growth regulator-free medium. In other part of study, the behavior of different cumin genotypes in direct regeneration pathways was studied. Both direct organogenesis and direct proliferation pathways were applicable to different cumin genotypes and regenerated plants were phenotypically normal. This study supports the feasibility of combined direct regenerations protocols from embryo and node of cumin in germplasm conservation by in vitro cloning and genetic improvement programs.

AN ALTERNATIVE PATHWAY FOR PLANT IN VITRO REGENERATION OF INSECTICIDAL TREE MELIA AZEDARACH L. DERIVED FROM THE INDUCTION OF SOMATIC EMBRYOGENESIS

A model for highly efficient method for plant regeneration from somatic embryos of chinaberry tree (Melia azedarach L.) was obtained. Mature seeds were cultured on Murashige and Skoog's medium supplemented with NAA/BA to induce embryogenic tissues. Embryo proliferation occurred with high level citokinin concentration (10 mg/L BAP) and germination occurred on free hormone medium or in BAP/AG 3 medium. Plants were regenerated from both, direct embryo formation and through an intermediary callus phase. Results showed 100% embryo conversion to plant. Regenerated plants were normal phenotypically and continued to grow after transfer to a greenhouse environment with 100% of surviving with high rate of growth.

Direct shoot regeneration from mature embryo as a rapid and genotype-independent pathway in tissue culture of heterogeneous diverse sets of cumin (Cuminum cyminum L.) genotypes

A rapid and one-step protocol for direct regeneration of shoots from cumin embryo explants has been developed. Embryo explants with shoot meristems were cultured on shoot regeneration medium for 15–22 d. After embryo culture, shoots were regenerated from the area adjacent to the region between the cotyledons and embryo axis within 2 wk, without any intermediate callus phase. Shoot proliferation and elongation were achieved on shoot regeneration medium without subculture. Among the different combinations of 6-benzylaminopurine, α-naphthaleneacetic acid (NAA), and indole-3-acetic acid (IAA) tested, 0.8 mgl−1 (4.3 μM) NAA in combination with 0.3 mgl−1 (1.71 μM) IAA in the B5 medium resulted in the most efficient direct shoot regeneration. No significant difference was detected for the number of regenerated explants when different heterogeneous endemic varieties were compared. This plant regeneration procedure was applicable to different cumin genotypes and regenerated plants were phenotypically normal.

Zeatin overcomes thidiazuron-induced inhibition of shoot elongation and promotes rooting in strawberry culture in vitro

SummaryThe effects of 0, 2, 4 or 8 µM thidiazuron (TDZ) and explant type on adventitious shoots regenerated on excised leaf disks, sepals (calyx) and petiole halves of greenhouse-grown ‘Bounty’ strawberry (Fragaria ananassa Duch.) were studied. A moderate concentration (2 – 4 µM TDZ) supported bud and shoot regeneration without an intermediary callus phase on young expanding sepals, or on leaf disks with the adaxial side touching the culture medium, or on petiole halves placed horizontally with their cut-sides in contact with the medium, all maintained for 14 d in the dark. Sepals proved more effective than leaf discs and petiole segments for regenerating shoots. Another experiment compared the effects of 0, 0.5, 1, 2 and 4 µM TDZ or zeatin on shoot proliferation and rooting of sepal-derived adventitious shoots. Media containing TDZ generally promoted more callus formation and suppressed elongation and rooting of shoots. Shoots proliferated and roots developed best when explants were cultured in medium supplemented with 1 or 2 µM zeatin. An in vitro cloning protocol that enables shoot proliferation and rooting of strawberry explants in the same medium is expected to be applied for commercial micropropagation.

Strawberry sepal: Another explant for thidiazuron-induced adventitious shoot regeneration

An efficient system to regenerate shoots on excised sepals (calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria x ananassa Duch.) was developed in vitro. Sepal cultures produced multiple buds and shoots without an intermediary callus phase on 2–4 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation. Young expanding sepals with the adaxial side touching the culture medium and maintained for 14 d in darkness produced the best results. In a second experiment, sepals proved more effective than the leaf discs and petiole segments for regenerating shoots. A third experiment compared the effects of six concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4 μM and zeatin at 2 and 4 μM) for elongation of sepal-derived adventitious shoots. The media containing TDZ generally promoted more callus formation and suppressed shoot elongation. TDZ-initiated cultures transferred into the medium containing 2–4 μM zeatin, produced usable shoots after one additional subculture. Shoots were rooted in vitro in the same medium used for shoot regeneration, but without any growth regulators. When transferred to potting medium, 85–90% of in vitro plantlets survived.

Efficient in vitro direct shoot organogenesis and regeneration of fertile plants from embryo explants of Bambara groundnut ( Vigna subterranea L. Verdc.).

An efficient protocol has been developed for direct shoot organogenesis from embryo axes derived from mature seeds of two different landraces of Bambara groundnut. Multiple shoots were initiated on several media containing different concentrations and combinations of benzylaminopurine (BAP) or thidiazuron (TDZ). Efficient regeneration occurred when the embryo axes were first plated for 6 days on a medium containing high concentrations of BAP (1 mg/l) and alpha-naphthaleneacetic acid (NAA, 1 mg/l) and then cut transversely and transferred onto a medium containing 1.5 mg/l BAP. Shoot regeneration frequency was 100% and from five to eight shoots per explant were obtained. The importance of using embryo explants and cytokinins in the culture media, with respect to controlling the development of a highly organogenic system, was demonstrated. Histological studies revealed that proliferating buds originated directly from the superficial layers of the explants without an intermediate callus phase. The regenerated shoots were rooted on a medium containing 1 mg/l NAA and then transferred to the greenhouse. Flow cytometric analyses and chloroplast counts of guard cells suggested that the regenerants were diploid. All were morphologically normal and fertile. The short duration, high efficiency and low frequency of somaclonal variation of this system make it well suited for wider biotechnological applications of Bambara groundnut-a neglected and under-utilized crop.

An efficient adventitious shoot regeneration system on excised leaves of micropropagated lingonberry (Vaccinium vitis-idaea L.)

SummaryAn efficient system to regenerate shoots in vitro on excised leaves of lingonberry (Vaccinium vitis-idaea L.) was developed. Leaf explants from shoot-proliferating cultures produced multiple shoots without an intermediary callus phase on zeatin (ZN)-containing shoot induction media within 3–4 weeks of culture initiation. Cultivars Regal and Splendor, and one clone from a natural stand in Estonia (ECL1), were used in the first experiment. Young expanding leaves with the adaxial side touching the culture medium, and maintained for 7 d in darkness, produced the best results. There were significant genotypic differences in adventitious shoot formation. A second experiment studied the effects of ten concentrations of three cytokinins: ZN at 5, 10, 20, 30 and 40 μM; 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ) at 0.1, 1, 5 and 10 μm; and 6-(γ-γ-dimethylallylamino) purine (2ip) at 25 μM were compared with leaf segments of different polarity in ‘ECL1’. Zeatin was found to be more effective than TDZ or 2iP as an inductive signal for regenerating many vigorous shoots. Zeatin induced multiple shoot formation at all concentrations tested, but maximum morphogenic response was observed at 20 to 30 μM. The media containing TDZ generally promoted more callus formation and suppresed shoot elongation. In a third experiment with the lingonberry cultivar Erntedank and the clone ‘ECL1’, a new medium developed for lingonberry shoot culture proved more effective than the modified Murashige and Skoog medium for regenerating shoots on leaf explants. Elongated shoots were excised and rooted directly on a 2 peat:1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid. Rooted plantlets were acclimatized under greenhouse conditions to evaluate somaclonal variation.

Vacuum Infiltration Based Agrobacterium Mediated Gene Transfer to Lentil (Lens Culinaris M.) Tissues

ABSTRACTA highly efficient Agrobacterium-mediated transformation system has been developed for lentils (Lens culinaris M.) using vacuum infiltration of cotyledonary nodes and nodal segments with Agrobacterium suspension. This procedure exhibits distinct advantages over those previously reported in lentils, in that it uses vacuum infiltration enhancive force to introduce Agrobacterium suspension into the highly regenerable cotyledonary node meristems or nodal segments, which in turn rapidly produce transgenic shoots without an intermediate callus phase. The efficiency of the system has been investigated using GUS histochemical assays and PCR amplification which were evidencing integration of the transgenes and superiority of this system over conventional Agrobacterium mediated procedures that has been so far applied for lentil transformation.