Direct Shoot Organogenesis Research Articles

Direct shoot organogenesis from in vitro-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl−1 IAA and 2 mgl−1 BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl−1 BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl−1 of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.

TDZ-induced direct shoot organogenesis and somatic embryogenesis on cotyledonary node explants of lentil (Lens culinaris Medik.)

An efficient and simple procedure for inducing high frequency direct shoot organogenesis and somatic embryogenesis in lentil from cotyledonary node explants (without both the cotyledons) in response to TDZ alone is reported. TDZ at concentration lower than 2.0 μM induced shoot organogenesis whereas at higher concentration (2.5-15 μM) it caused a shift in regeneration from shoot organogenesis to somatic embryogenesis. The cotyledonary node and seedling cultures developed only shoots even at high concentrations of BAP and TDZ, respectively. TDZ at 0.5 and 5.0 μM was found to be optimal for inducing an average of 4-5 shoots per cotyledonary node in 93 % of the cultures and 55 somatic embryos in 68 % of the cultures, respectively. The somatic embryos were germinated when transferred to lower TDZ concentration (0.5-1.0 μM). The shoots were rooted on MS basal medium containing 2.5 μM IBA. The plantlets were obtained within 8 weeks from initiation of culture and were morphologically similar to seed-raised plants. The possible role of stress in thidiazuron induced somatic embryogenesis is discussed.

In vitro micro-propagation of endangered ornamental plant-Neotchihatchewia isatidea (Boiss.) Rauschert

The ornamental plant, Neotchihatchewia isatidea, is an endangered species of Turkey and threatened by complete extinction in the future. Therefore, in vitro multiplication of this species can be valuable for commercial production and germplasm conservation. Immature embryos of N. isatidea were cultured for initiation on Murashige and Skoog medium (MS) supplemented with N6-benzylamino-purine (BAP) and -naphthaleneacetic acid (NAA). Shoot primordia were visible within 5 - 6 weeks and the shoot primordia later developed into normal shoots 10 - 12 weeks after the culture initiation on calli developed from immature embryos. Shoot tips were also excised from developed plantlets for direct shoot organogenesis and cultured on MS shoot induction medium supplemented with BAP (0.5, 1.0 and 2.0 mg/l), kinetin (KIN) (0.5, 1.0 and 2.0 mg/l) and thidiazuron (TDZ) (0.05, 0.10 and 0.50 mg/l). Direct multiple shoots from shoot tips developed in most media tested. High shoot multiplication (3.73), high rooting (53 %) number of root per shoot (3.66) and survival ratio (46.6 %) were achieved.

Biochemical and histological changes associated with in vitro responses in sunflower cotyledonary explants

In vitro shoot regeneration from sunflower cotyledonary explants can be obtained in the presence of kinetin and indole-3-acetic acid. In contrast, callus prolif- eration is obtained in the presence of 2,4-dichlorophenoxy- acetic acid on culture medium. The purpose of this study was to investigate changes in protein profiles during callus and shoot development from cotyledonary explants and to correlate them with ontogenic stages during in vitro culture. Cotyledons cultured in the presence of 2,4-dichlorophe- noxyacetic acid produced friable callus as a result of early division of parenchymatic cells associated with the vascular bundles of the explant. The callogenic ability was indepen- dent of the cotyledonary region used as starting explant. Direct shoot organogenesis was observed from the same type of cells growing in culture media supplemented with kinetin and indole-3-acetic acid. In this case, the regener- ation potential varied among regions from which the explants were obtained. Protein profiles revealed differ- ences associated with shoots or callus developmental programs. A 27-kDa polypeptide was uniquely detected in the explants undergoing shoot organogenesis. The amount of this polypeptide during the first 4 d of culture increased and was followed by the appearance of meristematic centers in histologically analyzed samples. This polypeptide could be used as a specific marker for in vitro shoot development in this species.

Induction and comparison of different in vitro morphogenesis pathways using embryo of cumin (Cuminum cyminum L.) as a model material

In this study, using cumin embryo as explant and manipulating plant growth regulators (PGRs) in regeneration medium, the main in vitro morphogenesis pathways including direct shoot organogenesis, direct somatic embryogenesis, indirect somatic embryogenesis, and indirect shoot organogenesis were obtained. The effects of PGRs, subculture, and light on the induction and progression of different pathways were studied in detail. Direct shoot organogenesis occurred on the meristematic zone, while direct somatic embryogenesis was observed on hypocotyl part of cumin embryo (more differentiated part). Application of BAP (0.1 mgl−1) was the sole triggering factor for induction of callus and indirect regeneration pathways. Exogenous IAA played the central role in the direct somatic embryogenesis pathway; however, the combined effects of IAA and NAA along with the high endogenous cytokinin level resulted in direct shoot organogenesis. Subculturing revealed accelerating effects on direct somatic embryogenesis pathway and callus formation. Conversely, subculturing had negative effect on direct shoot organogenesis pathway. In certain combinations of PGRs, like 0.4 mgl−1 IAA + 0.4 mgl−1 NAA, co-induction and co-regeneration of different pathways were observed. Investigation of genotype dependencies of different pathways showed that direct pathways are more genotype-dependent, stable, and faster than indirect pathways. This research presents the embryo of cumin as a convenient model material for induction and comparison of different morphogenesis pathways.

Organogenesis plant regeneration from leaf explants of Exacum Styer Group

Exacum Styer Group plantlets were regenerated through direct organogenesis from leaf explants. Four genotypes were evaluated on MS media supplemented with combinations of BA (0, 0.44, 2.22, 4.44, or 8.88 μM) and NAA (0, 0.05, 0.54, or 2.69 μM) for direct shoot organogenesis without an intervening callus phase. Regression analyses were used to analyze and interpret the data. There were significant genotype, media, and genotype × media interactions for several variables. Genotypes 01-09-01 and 01-37-61 had the highest number of shoots per explant across media (10.2 and 6.6, respectively) while the 4.44 μM BA plus 0.54 μM NAA treatment induced the greatest number of shoots among the genotypes evaluated.

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.

SETTING UP TISSUE CULTURE TECHNIQUES FOR REGENERATION OF APRICOT TRANSGENIC SHOOTS IN A TRANSFORMATION PROGRAM FOR RESISTANCE TO PLUM POX VIRUS

For recalcitrant woody species, such as apricot, one of the most critical steps of genetic transformation is inducing plant regeneration specifically from those explant cells that are (or become, in response to culture conditions) competent for stable transgene integration. In previous trials we were able to obtain regeneration from both mature tissues and zygotic embryos of various apricot cultivars with methods that, nevertheless, proved to be unsuitable for recovering transgenic plants, when applied to Agrobacterium-mediated transformation. Using this system, the shoots and somatic embryos that promptly differentiated from the explant tissues neither revealed GUS activity nor survived kanamycin selection, while callus masses proximally associated to them were eventually transformed. On the other hand, when we applied higher hormone levels than those required for direct shoot organogenesis to stimulate first proliferation of kanamycin-resistant cells, then their induction to morphogenesis, we achieved higher frequencies of stable transgene integration but recovered callus lines instead of normally developed shoots. The present work presents an update on the tissue culture methods under evaluation in our laboratory for indirect shoot regeneration. In order to shorten the callus proliferation phase with respect to our past experiments, we modified the in vitro culture conditions applied to leaf and stem explants of micropropagated cultivars ('Boreale', 'San Castrese', 'Sungiant', and 'Vitillo') inoculated with A. tumefaciens EHA101 pGA482GG/PPV-CP-33. Additionally, different substrates and subculture periods have been tested for shoot development from the transformed callus lines obtained from previous experiments. The newly-inoculated explants, consistent with our past results, yielded no transgenic shoots, but did produce kanamycin-resistant cell proliferation. Some parameters modified in the present tests promoted a slow callus hardening process and the production of bud-like structures from both the more recently obtained and the older transgenic material.

Direct shoot organogenesis and plant regeneration in Fortunella crassifolia

An efficient in vitro regeneration system in kumquats (Fortunella crassifolia Swingle) was established. Explant types and orientations, concentrations and combinations of plant growth regulators were evaluated for their influences on efficiency of plant regeneration. It was found that the optimum explant and its orientation was epicotyl planted vertically with upper part upward, and a shoot regeneration frequency of 1.48 shoots per explant was obtained on Murashige and Skoog (1962; MS) medium supplemented with 22.19 μM 6-benzyladenine (BA). A rooting percentage as high as 74 % was obtained on 1/2 MS supplemented with 0.54 μM 1-naphthaleneacetic acid (NAA), 9.29 μM kinetin (KN), and 0.5 g dm-3 activated charcoal.

In vitro propagation of Cleome spinosa (Capparaceae) using explants from nursery-grown seedlings and axenic plants

Two independent experiments were performed to establish micropropagation of Cleome spinosa from stem segments. In the first experiment, direct shoot organogenesis on hypocotyl explants from 2-mo.-old nursery-grown seedlings was obtained on Murashige and Skoog medium with different combinations of benzyladenine (BA) and 6-furfurylaminopurine, added either individually or in combination. Best proliferation rates occurred in the presence of 2.2 and 4.4 μM BA and the highest mean number of shoots was produced in response to 4.4 μM BA. In the second experiment, regeneration via direct organogenesis was also obtained from nodal and internodal segments of axenic plants cultured in the presence of BA (4.4 and 8.8 μM) in association with indole-3-acetic acid (IAA) (0.57 and 1.14 μM). Internodal explants were the most responsive on all media tested. The best mean number of shoots per explant was achieved on medium with 4.4 μM BA in association with 0.57 μM IAA. Histological studies of the globular structures formed at the apical portion of the explants revealed direct shoot regeneration and adventitious shoot differentiation from meristematic centers around the vascular bundles of the primary regenerants. All shoots elongated and rooted on MS0 medium. The acclimatization rates ranged between 70 and 84%. Plants reached to maturity and flowered 4 mo. after transfer to ex vitro conditions.

Production of the first transgenic cassava in Africa via direct shoot organogenesis from friable embryogenic calli and germination of maturing somatic embryos

The impact of cassava transformation technologies for agricultural development in Africa will depend largely on how successfully these capabilities are transferred and adapted to the African environment and local needs. Here we report on the first successful establishment of cassava regeneration and transformation capacity in Africa via organogenesis, somatic embryogenesis and friable embryogenic callus (FEC). As a prerequisite for genetic engineering, we evaluated six African cassava genotypes for the ability of a) induction of FEC b) hygromycin sensitivity and c) T-DNA integration potential by different Agrobacterium strains. FEC was induced in genotypes TMS 60444, TME 1 and TMS 91/02327. Potential tissues for FEC formation were induced in TMS 91/02324, TME 12 and TME 13. Pure and proliferating FEC was obtained and maintained only in TMS 60444. FEC growth and shoot organogenesis were completely suppressed when hygromycin was used at a concentration of 20 mg/l in all tissue types and genotypes. With somatic cotyledons, statistically significant differences (p
0.05) were observed between Agrobacterium strains and genotypes with respect to T-DNA transfer efficiency. Using somatic cotyledons, TME 8 was found to be the most amenable to transformation with maximum blue spots per GUS-positive explants, and Agrobacterium GV3101 proved to be superior to EHA105, LBA4404, and AGl-1 for T-DNA transfer based on transient assays with a reporter gene (GUS). With FEC, Agrobacterium LBA4404 was superior to other strains. This study also identified EHA105 as a new vir helper strain to recover transgenic cassava plants. PCR and Southern hybridization of genomic DNA of the hygromycin-resistant cassava plants to a hpt probe confirmed the integration of hpt with integration events varying between 1 and 2 insertions. The benefit of combining the FEC and shoot organogenesis systems for recovering transgenic cassava plants is described. The contributions of this report to enhancing the development and deployment of genetic engineering of cassava for agricultural biotechnology development in Africa are discussed.

Developmental and hormonal regulation of direct shoot organogenesis and somatic embryogenesis in sugarcane (Saccharum spp. interspecific hybrids) leaf culture

Rapid and efficient in vitro regeneration methods that minimise somaclonal variation are critical for the genetic transformation and mass propagation of commercial varieties. Using a transverse thin cell layer culture system, we have identified some of the developmental and physiological constraints that limit high-frequency regeneration in sugarcane leaf tissue. Tissue polarity and consequently the orientation of the explant in culture, size and developmental phase of explant, and auxin concentration play a significant role in determining the organogenic potential of leaf tissue in culture. Both adventitious shoot production and somatic embryogenesis occurred on the proximal cut surface of the explant, and a regeneration gradient, decreasing gradually from the basal to the distal end, exists in the leaf roll. Importantly, auxin, when added to the culture medium, reduced this spatial developmental constraint, as well as the effect of genotype on plant regeneration. Transverse sections (1-2 mm thick) obtained from young leaf spindle rolls and orienting explants with its distal end facing the medium (directly in contact with medium) are critical for maximum regeneration. Shoot regeneration was observed as early as 3 weeks on MS medium supplemented with alpha-naphthalenencetic acid (NAA) and 6-benzyladenine, while somatic embryogenesis or both adventitious shoot organogenesis and somatic embryogenesis occurred on medium with NAA and chlorophenoxyacetic acid. Twenty shoots or more could be generated from a single transverse section explant. These shoots regenerated roots and successfully established after transplanted to pots. Large numbers of plantlets can be regenerated directly and rapidly using this system. SmartSett, the registered name for this process and the plants produced, will have significant practical applications for the mass propagation of new cultivars and in genetic modification programs. The SmartSett system has already been used commercially to produce substantial numbers of plants of orange rust-resistant and new cultivars in Australia.

In vitro Direct Shoot Organogenesis and Regeneration of Plantlets from Leaf Explants of Sentang (Azadirachta excelsa)

In vitro Direct Shoot Organogenesis and Regeneration of Plantlets from Leaf Explants of Sentang (Azadirachta excelsa)

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.

An improved system for the in vitro regeneration of Thapsia garganica via direct organogenesis – influence of auxins and cytokinins

An improved protocol for mass multiplication directly from leaf material of Thapsia garganicawas developed. Using factorial experimentation, auxins (NAA, IAA, 2,4-D) and cytokinin (BA, kinetin) combinations at 0–2 mg l−1 added to Murashige and Skoog (MS) medium with 30 g l−1 sucrose and 8 g l−1 agar (pH 5.8) were tested for their effect on direct regeneration on leaflet and petiole explants. Of the media tested, the 0.5:1.5 NAA:BA medium was comparable for direct shoot organogenesis to the 2 mg l−1 kinetin supplemented medium. However, when shoots were multiplied on these media, the 2 mg l−1 kinetin without auxins was most effective as it kept the percentage of callus-derived plantlets to 3% and the number of hyperhydric shoots were minimal at a frequency of 2% compared to 25% on the 0.5:1.5 NAA:BA medium. The 2 mg l−1 kinetin medium induced adventitious bud formation in 36% of the explants after 30 days. When the cultures were transferred to the same medium for multiplication, an average of six shoots (4.3 cm) were derived from each shoot base. Other combinations resulted in callus formation that either preceded shoot production or occurred together with adventitious shoot induction; whereas the 2 mg l−1 medium resulted solely in adventitious buds that readily converted and elongated to shoots. On the 0.5:1.5 NAA:BA medium which tended to induce hyperhydric shoots in culture, agar (0.8, [w/v]) (15% hyperhydric plantlets) was more useful in maintaining a high health status in regenerating plantlets than gellan gum (Gelrite®; 0.25, [w/v]) (60% hyperhydric plantlets). Although rooting in vitro was difficult, 58% of the propagules were successfully acclimatized when plants were exposed to fungicidal solutions as pre- and post-acclimatization treatments. A comprehensive protocol that allows for a reduction in mortality due to damping-off diseases during ex vitro transplantation of the in vitro-derived T. garganica plantlets is reported. The acclimatization procedure presented here is potentially suited to other umbelliferous species where fungal rots hamper ex vitro establishment.

(405) Regeneration of Venus Fly Trap (Dionaea muscipula Ellis) from Leaf Culture

Dionaeamuscipula Ellis commonly known as Venus fly trap is an important carnivorous plant with medicinal importance. It contains certain secondary metabolites like naphthoquinones and is used in anti-aid and anti-cancer drugs and other medicines like Cornivora. Increasing interest and use as an ornamental and medicinal plant, and dietary supplement have put it in an endangered state. Development of in vitro techniques for the preservation of germplasm that is on the brink of extinction is highly demanded. A regeneration protocol for the multiplication and micropropagation of Dionaeamuscipla Ellis was established. In vitro regeneration potential of leaf explants in different concentrations and combinations of plant growth substances was investigated in this study. Seeds were grown and leaf disc explants were excised and cultured under aseptic conditions on nutritional medium containing half strength Murashige and Skoog (MS) mix with combinations of 1.0–20.0 μm BA, 2.5.0 μm IBA, 1.0–10.0 μm 2iP and 0.1–0.5μm TDZ. The cultures were kept in growth cabinet with cool white light (40–60 μmol·m-2·s-1) under 16-h photoperiod. Regeneration was recorded after 60 days with the intervals of 15 days based on the degree of shoot organogenesis and somatic embryogenesis. 1/2 MS + 0.1 TDZ appeared to be efficient for somatic embryogenesis and simple MS for direct shoot organogenesis. 1/2 MS combined with 2iP appeared to be efficient for regeneration either by direct shoot organogenesis or by somatic embryogenesis. Plants were rooted well in Cape Cundew medium. These investigations will aid in the development of a model system for clonal mass propagation and in vitro regeneration of Dionaeamuscipla Ellis.

Direct shoot organogenesis on shoot apex from seedling explants of Capsicum annuum L.

In vitro direct multiple shoot formation from seedling explants of Indian high pungent varieties of Capsicum annuum cv. Arka Abhir (AA) and Arka Lohit (AL) was successfully obtained. We were able to induce regeneration potency in these varieties by inverting the explant. Aseptically grown seedling explants with decapitated roots, apical meristem and cotyledonary leaves were inoculated in an inverted position in bud induction medium comprising of Murashige and Skoog's basal medium supplemented with 2-( N-morpholine) ethanesulphonic acid (MES) buffer along with 26.63 μM benzyl adenine (BA), 2.28 μM indole-3-acetic acid (IAA) and 10 μM silver nitrate. Profuse shoot bud induction with 20–25 shoot buds per explant was obtained. Supplementation of phloroglucinol in the bud induction medium resulted in 17 and 18% enhancement in bud induction response in Arka Abhir and Arka Lohit variety, respectively on the inverted hypocotyls. Auxin transport inhibitor tri-iodo benzoic acid (TIBA) in the bud induction medium resulted in induction of buds in a shorter period of 40–45 days when compared to bud induction (BI) medium which takes 55–65 days for bud induction. These buds were transferred to MS medium containing 2.8 μM gibberellic acid and 10 μM silver nitrate resulting in elongation of shoot buds. Transfer of shoots to MS basal medium induced rooting to produce plantlets. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. annuum tissues.

Agrobacterium-mediated transformation of faba bean (Vicia faba L.) using embryo axes

A system for the production of transgenic faba bean by Agrobacterium-mediated transformation was developed. This system is based upon direct shoot organogenesis after transformation of meristematic cells derived from embryo axes. Explants were co-cultivated with A. tumefaciens strain EHA105/pGlsfa, which harbored a binary vector containing a gene encoding a sulphur rich sunflower albumin (SFA8) linked to the bar gene. Strain EHA 101/pAN109 carrying the binary plasmid containing the coding sequence of a mutant aspartate kinase gene (lysC) from E. coli in combination with neomycinphosphotransferase II gene (nptII) was used as well. The coding sequences of SFA8 and LysC genes were fused to seed specific promoters, either Vicia faba legumin B4 promoter (LeB4) or phaseolin promoter, respectively. Seven phosphinothricin (PPT) resistant clones from Mythos and Albatross cultivars were recovered. Integration, inheritance and expression of the transgenes were confirmed by Southern blot, PCR, enzyme activity assay and Western blot.

Direct shoot organogenesis from needles of three genotypes of Sequoia sempervirens

Using in vitro-grown needles of Sequoia sempervirens (D. Don.) Endl., direct shoot organogenesis was induced. The effects of three genotypes and two cytokinins, N6-benzyladenine (BA) and N-benzyl-9 (2-tetrahydropyranl) adenine (BPA), in combination with 2,4-D were investigated. Among tested cytokinins, BPA produced the highest frequency of shoot organogenesis from all three genotypes tested. Adventitious shoots were induced directly from explants without intervening callus within 5 weeks following incubation. Shoots were elongated on a 1/2 Wolter and Skoog (WS) medium supplemented with activated charcoal but without growth regulators. Later, elongated shoots were transferred to a 1/4 WS medium, but without activated charcoal and free of plant growth regulators to promote continued shoot growth. These shoots rooted spontaneously.

In situ stress measurement and its application for hydro-electric projects ¿ an indian experience in the Himalayas

We propose a new numerical method for calculating 2D fractal dimension (DF) of a surface. This method represents a generalization of Higuchi’s method for calculating fractal dimension of a planar curve. Using a family of Weierstrass–Mandelbrot functions, we construct Weierstrass–Mandelbrot surfaces in order to test exactness of our new numerical method. The 2D fractal analysis method was applied to the set of histological images collected during direct shoot organogenesis from leaf explants. The efficiency of the proposed method in differentiating phases of organogenesis is proved.