Volume 4 Number 1 2010

CONTENTS AND ABSTRACTS

Chris Ojiewo (Japan/Tanzania), Yasutaka Kubo, Kenji Murakami, Masaharu Masuda (Japan) Comparative Analysis of Differential Gene Expression in Wild-type and ¹²C⁵⁺ Ion Beam-induced Abnormal Flower Mutant of Solanum villosum by Tomato cDNA Macroarray (pp 1-7)

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Original Research Paper: The differential expression of genes in an abnormal floral organ mutant of Solanum villosum, T-5, was compared with those in wild-type (W-T) plants at the pre-anthesis bud stage using a tomato cDNA macroarray. Genes whose expression was three-fold higher or lower in the mutant than in wild-type plants were considered to be up- or down-regulated, respectively. Of 11520 genes, differential expression of genes was observed in a total of 122 genes out of which 45.9% were down-regulated while 54.1% were up-regulated in the mutant. The functional distribution of differentially expressed genes included cellular biological processes potentially associated with floral patterning such as regulation of metabolism, transcription, cellular communication and signal transduction mechanisms, systemic interaction with the environment and tissue/organ differentiation based on the annotated catalogue of Munich Information Center for Protein Sequences (MIPS). Down-regulated genes with potential effects on floral organ specification included homologues of Sepallata 1 (SEP1), Agamous (AG), HUA1 and Circadian Clock Associated 1 (CCA1). With the identification the genes with putative transcription factor activity in the control of T-5 floral organ identity, we have moved closer to a complete understanding of the underlying factors and the culprit gene responsible for the differences in W-T and T-5 mutant.

Omar Aldahhak, Zaid Salim (Syria), Jaime A. Teixeira da Silva (Japan), Ahmad M. Abdul-Kader (Syria) In Vitro Approach to the Multiplication of a Halophyte Species Forage Shrub Atriplex halimus L. and in Vitro Selection for Salt Tolerance (pp 8-14)

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Original Research Paper: A successful and detailed in vitro multiplication system for micropropagation of the forage shrub Atriplex halimus L. has been developed. Explants excised from adult shrubs were surface-disinfected before being placed onto Murashige and Skoog (MS) basal medium containing a combination of plant growth regulators (PGRs) at different concentrations: N⁶-benzyladenine (BA) or kinetin at 2.22 or 4.44 µM each with indole-3-butyric acid (IBA) at 0.49 µM and gibberellic acid (GA₃) at 0.58 µM. A 7.2-fold multiplication rate was achieved every 4 weeks on MS medium supplemented with 4.44 µM BA, 0.49 µM IBA and 0.58 µM GA₃. Rooting was achieved with 73% efficiency within 2-4 weeks on agar-gelled MS basal medium free of PGRs. Rooted plantlets were gradually acclimatized to field conditions over 5-6 weeks with 65% efficiency. MS medium was supplemented with increasing concentrations of NaCl ranging between 25 and 1000 mM to study the effects on multiplication and to select high salt-tolerant clones in vitro. In vitro shoots could tolerate up to 600 mM NaCl with optimal growth at 200 mM, while higher concentrations of NaCl affected growth negatively. Growth and shoot number decreased with increasing NaCl concentration; all plantlets died at 1000 mM NaCl. A few clones survived high concentrations of NaCl (600 mM) which are being propagated under the same stress conditions for further studies. This method has the potential to produce masses of plantlets within a short time to expand its cultivation in dry and saline areas. Doing so would contribute to alleviating desertification and providing fodder for livestock, mainly during the dry season.

Mohamed Reda Abd Almegid Abd Alhady, Mohamed Mohamed Abd Alla, Ghada Abd El-moneim Hegazi, Mahdia Farid Gabr. (Egypt) Rapid Propagation of Periploca angustifolia Labill. by Tissue Culture (pp 15-18)

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Original Research Paper: Periploca angustifolia Labill. (Asclepiadaceae) is an extremely rare fodder shrub native to Egypt which is being severely affected by habitat loss and overgrazing due to its high palatability to animals. Tissue culture of this species has not been previously reported and may be a method for its conservation and propagation as it is heavily overexploited. An efficient and rapid method for micropropagation of P. angustifolia was developed by nodal stem segments collected from mature shrubs in the wild. Nodal explants were established on Murashige and Skoog (MS) basal medium containing 3% sucrose supplemented with different concentrations of 6-benzylaminopurine (BAP) (0.2, 0.5 and 1.0 mg l^-1) in combination with β-naphthalene acetic acid (NAA) (0.1 and 0.2 mg l^-1). Shoots could be multiplied on MS medium containing 3% sucrose supplemented with BAP (0.5-2.0 mg 1^-1) and N6-(2-isopentenyl) adenine (2iP) (0.5 mg 1^-1). The maximum number of proliferated shoots was obtained on MS medium containing 3% sucrose supplemented with 2 mg l^-1 BAP + 0.5 mg l^-1 2iP. Indole-3-butyric acid (IBA) gave a better rooting response than NAA. Most (70%) shoots rooted on half-strength MS medium containing 3% sucrose and 2.0 mg 1^-1 IBA to obtain complete plantlets. Eighty percent of the in vitro rooted plantlets were successfully hardened in the soil under greenhouse conditions. The use of this method appears to be a promising approach for population reinforcement and for in vitro preservation programs of threatened and rare populations.

Saikat Gantait, Nirmal Mandal, Somnath Bhattacharyya, Prakash Kanti Das (India) Sustainable in Vitro Propagation and Clonal Fidelity in Strawberry (pp 19-25)

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Original Research Paper: An efficient protocol was developed for sustainable mass multiplication of strawberry (Fragaria × ananassa Duch cv. ‘Chandler’) through multiple shoot induction. Shoot buds were most successfully induced from runner tips, when these were cultured on Murashige and Skoog (MS) medium supplemented with 0.1 mg l^-1 α-naphthalene acetic acid and 1 mg l^-1 6-benzylaminopurine. Maximum shoot multiplication and proliferation occurred on MS medium with 2 mg l^-1 kinetin. MS basal medium with 0.5 mg l^-1 indole-3-acetic acid and 2 g l^-1 activated charcoal proved to be the best for root induction and elongation from separated shoots. Autoclaved sand and soil with intermittent water spraying could optimize the primary acclimatization of in vitro generated plantlets. A pot mixture combined with sand, soil and farm yard manure (1: 1: 1 v/v) resulted in the acclimatization of 92% of plantlets. To prove the genetic uniformity of propagules, in vitro generated clones were DNA fingerprinted using selected ISSR primers.

Gulzar S. Sanghera, Manjit S. Gill, Satbir S. Gosal (India) Optimization of Shoot Tip-Based in Vitro Plant Regeneration in Cotton (Gossypium spp.) (pp 26-30)

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Original Research Paper: An in vitro regeneration system in cotton has been optimized from explants comprising pre-existing shoot apices of aseptically grown seedlings. This system has been found to be useful with a variety of genotypes (arborium and hirsutum). Shoot regeneration among genotypes on different media ranged from 69 to 80%. The age of explants showed a significant effect on shoot tip regeneration. On an average, 5 day-old shoot tips recorded highest regeneration of 76% as compared to 3 or 9 day old seedlings on ½ MS medium + 6% sucrose. The genotypic differences for regeneration rate were significant, indicating that the regeneration of shoot tips was genotype-dependent. Rooting efficiency between genotypes ranged from 60 to 67% was not significantly different indicated that rooting efficiency is genotype independent under in vitro conditions. However, root induction was greatly influenced by media composition as it varied from 55 to 72%. Half MS + 0.05 mg/l α-naphthalene acetic acid medium recorded the highest percentage of root induction (72%) closely followed by half MS (68%). Root and shoot formation was observed in all genotypes. After plantlet formation, in vitro-raised plantlets were shifted to test tubes containing a little cotton soaked with a small amount of water for two weeks and the water from these tubes was changed daily. The hardened plantlets were then transferred to small polythene bags filled with sand: soil mixture in the greenhouse and finally transferred to earthen pots in glasshouse. These plants grew into healthy green plants and reached maturity.

H. P. Shilpashree, V. Ravishankar Rai (India) Effect of Plant Growth Regulators on Callus Formation, Plant Regeneration and Hypericin Production in Hypericum mysorense Hyne (pp 31-36)

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Original Research Paper: Hypericum mysorense Hyneis a pharmaceutically important medicinal plant found in Western Ghats of India. In this study a new system was developed for in vitro plant regeneration through callus formation and hypericin production. Leaf explants were inoculated onto MS (Murashige and Skoog) medium supplemented with different concentrations and combinations of 6-benzyl adenine (BA), kinetin (KIN), α-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxy acetic acid (2,4-D). Highest callus proliferation was observed when MS medium contained BA and NAA (1.0 mg/L each). Best shoot induction and multiplication was recorded on MS medium supplemented with 1.0 mg/L BA. A significant difference was recorded in average number and length of shoots/explant among the different concentrations of BA and KIN investigated. Roots were induced on MS media in the presence (0.5 mg/L) and absence of indole-3-acetic acid (IAA). Regenerated plantlets were successfully established in soil with a 98% survival rate. The concentration of hypericin was evaluated in different tissues of in vitro and ex vitro grown plants by using high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). Hypericin levels were higher in callus (5.0 µg/g, w/v) and leaves (4.1 µg/g, w/v) of in vitro plantlets than those of ex vitro leaf samples (3.1 µg/g, w/v). Results confirmed that plant growth regulators play an important role in the production of hypericin under controlled in vitro conditions. This is the first ever report on a rapid plant regeneration system for H. mysorense and provides an avenue for conservation strategies and phytomedicine production.

Kambaska Kumar Behera, Biswanath Bhunia (India), Jaime A. Teixeira da Silva (Japan), Santilata Sahoo (India) Plantlet Regeneration of Potato Yam (Dioscorea bulbifera L.) through in Vitro Culture from Nodal Segments (pp 37-41)

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Original Research Paper: Nodal vine segments from 6-week-old plants of Dioscorea bulbifera L. were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) and kinetin (Kn) together with α-naphthaleneacetic acid (NAA). Explants cultured in MS basal medium supplemented with 2.0 mg/l Kn + 1.0 mg BAP + 0.5 mg/l NAA showed highest number (8.5 ± 0.51) of multiple shoots. When excised, shoots raised in vitro were inoculated onto half-strength MS basal media supplemented with 2.0 mg/l NAA, where rooting was profuse (5.8 ± 0.39 roots per plantlet). Rooted shoots were transplanted to the greenhouse for hardening and; survival was 90% in field conditions without any visible morphological variation.

Ananya Das, Shyamal Ghose, Anjan Bhattacharyya, Animesh K. Datta (India) In Vitro Biogeneration of Alkaloids and Withanolides in Withania somnifera (L.) Dunal (Solanaceae) var. ‘Poshita’ and ‘Jawahar 22’ (pp 42-46)

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Original Research Paper: Withania somnifera (L.) Dunal (Family: Solanaceae; common name-Aswagandha, a plant species with immense therapeutic uses) var. ‘Poshita’and ‘Jawahar 22’ (recommended varieties) were assessed for their chemical contents (amount of total alkaloids and withanolide was estimated; withaferin A and withanolide A content were quantified by HPLC) under in vitro culture conditions in 3 different stages of callus development (stage I – undifferentiated callus, stages II and III – differentiated callus) on MS medium with different hormonal (BA, kin, 2,4-D, IAA, IBA) combinations using epicotyl, shoot tip and leaf explants with the objective of developing a suitable protocol which ensure production of specific stipulated amount of secondary metabolites in a quick span of time. Results obtained were discussed. Stage III of callus development yielded the maximum amount of secondary metabolites. The yield of alkaloids, withanolides and withaferin A were maximum in leaf explants while that of withanolide A was highest in cultures initiated from epicotyl explants, although the medium composition varied for each variety.

Parvatam Giridhar, Krishnaswamy Subbarayanakoppalu Sowmya, Akula Ramakrishna, Gokare Aswathanarayana Ravishankar (India) Rapid Clonal Propagation and Stevioside Profiles of Stevia rebaudiana Bertoni (pp 47-52)

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Original Research Paper: Shoot tips of Stevia rebaudiana Bertoni were cultured on Murashige and Skoog (MS) or B5 medium, supplemented with 6-benzyladenine (BA), indole-3-butyric acid (IBA) or a-naphthalene acetic acid (NAA) in different combinations. A maximum of 28 shoots per shoot tip explant were produced on B5 medium supplemented with 4.44 µM BA and 0.80 µM NAA. Significantly more shoots could be obtained from in vitro shoot tips of Stevia by repeating the cycle. Elongation of microshoots was significant on B5 medium devoid of plant growth regulators. MS medium containing 0.05 µM-0.25 µM gibberellic acid (GA₃) led to elongation of shoots up to 10-12 cm in length with 7-8 nodes each. In vitro leaves derived from primary shoots could regenerate 3-4 shoots on the same medium. In vitro rooting of microshoots (~4 cm in length) was efficient on MS basal medium with shoots 12-13 cm long having 5 nodes and 11-12 roots in one month. After hardening in micropots for one month 80% of the rooted plants survived. In another study, incorporation of 13.62 µM thidiazuron (TDZ) in modified MS medium induced 11-12 multiple shoots from shoot tip explants when inoculated in reverse polarity i.e. shoot tips in downward direction (inverted mode). Both these methods could be useful for mass multiplication of S. rebaudiana. The stevioside content of both in vitro leaves and hardened three-months-old ex vitro plants was analysed by HPLC. The highest stevioside content (6.72% steviolbioside and 0.11% rebaudioside-A on a dry weight basis) were found in ex vitro and in vitro leaves, respectively.

M. Naeem, Mohd. Idrees, Tariq Aftab, M. Masroor A. Khan, Moinuddin (India) Changes in Photosynthesis, Enzyme Activities and Production of Anthraquinone and Sennoside Content of Coffee Senna (Senna occidentalis L.) by Triacontanol (pp 53-59)

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Original Research Paper: Coffee senna plants grown in soil-containing pots were sprayed with five concentrations of TRIA [10^-0 (control), 10^-8, 10^-7, 10^-6 and 10^-5 M] at 15-day intervals. TRIA is a well known potent plant growth-promoting substance for many agricultural and horticultural crops. Keeping the importance of this medicinal plant in mind, a hypothesis was designed to determine whether foliar sprays of TRIA could augment crop productivity, photosynthesis, activities of enzymes, and the production of anthraquinone and sennoside content of coffee senna. The plant fresh and dry weights, total chlorophyll and carotenoid content, nitrate reductase activity, carbonic anhydrase activity, and leaf -N, -P, -K and -Ca contents were analyzed at 120, 270 and 300 days after sowing (DAS). Net photosynthetic rate, transpiration rate and stomatal conductance were measured only at 270 DAS. The seed-protein content and anthraquinone and sennoside contents were analyzed at harvest (330 DAS). Depending on the observed data, we conclude that a foliar spray of 10^-6 M TRIA significantly stimulated most of the studied attributes. A spray of 10^-6 M TRIA increased seed-yield and seed-protein content by 44.92 and 13.40%, respectively when compared to untreated plants. TRIA also stimulated the production of anthraquinone and sennoside contents compared to control plants.

Mohamed H. Al-Whaibi, Manzer H. Siddiqui, Abdullah Al-Amri, Mohammed O. Basalah (Kingdom of Saudi Arabia) Performance of Faba Bean under Calcium and Gibberellic Acid Application (pp 60-63)

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Original Research Paper: The main objective of the present studies was to determine the best dose of calcium (Ca⁺²) in Experiment 1, and in Experiment 2 aimed to test whether the growth and photosynthetic pigments of faba bean (Latin name) cv. ‘TARA’ could be enhanced by inclusion of GA₃ in the basal treatments containing Ca⁺². In Experiment 1, application of Ca⁺² significantly enhanced the almost growth and photosynthetic pigments enzyme carbonic anhydrase (CA) activity. Among the treatments, T₄ (60 mM Ca⁺²) proved best. Treatment T₄ significantly increased plant height, shoot FW, shoot DW, root length, root FW, root DW and root number, relative water content and chlorophyll (Chl) a, b, total Chl, anthocyanin and CA activity compared to the control. In Experiment 2, application of Ca⁺² with GA₃ significantly enhanced almost all growth parameters. Among the treatments, 20 mM Ca⁺² (T₃) with 10^-6 M GA₃ gave the maximum value for almost all parameters studied compared to the control as well as the application of GA₃ (10^-6) alone. The application of Ca⁺² along with GA₃ more efficiently ameliorates the growth and photosynthetic capacity of faba bean than Ca⁺² alone.

Emilio Mendoza-de Gyves, Maurizio Enea Picarella, Fabrizio Ruiu (Italy), Jaime A. Teixeira da Silva (Japan), Youssef Rouphael (Lebanon), Rosario Muleo, Eddo Rugini (Italy) Inhibition of Agrobacterium tumefaciens Growth by Silver Nitrate (pp 64-67)

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Original Research Paper: Silver nitrate has been found to enhance plant regeneration frequency probably due to its anti-ethylene activity. It has also been found to be a potential inhibitor of Agrobacterium tumefaciens growth after co-cultivation due to its bactericidal properties. A. tumefaciens strain GV2260, harboring a plasmid with the marker gene neomycin phosphotransferase II (nptII) and the chimeric gene Def H9-iaaM was used to assess the extent of susceptibility to AgNO₃. Bacterial growth curves were generated using turbidimetric measurements of bacterial density to evaluate bacterial response in the presence of different AgNO₃ concentrations. At low initial cell densities (OD₆₀₀ = 0.01 and 0.03) bacteria completely stopped growing at 8 and 10 mg l^-1 of AgNO₃, respectively while at the initial cell density of OD₆₀₀ = 0.1, silver ions strongly retarded cell growth at 25 mg l^-1; however, after 48 hrs they continued to grow, although at very slow rates. Also, bacterial fitness was assessed in the presence of kanamycin (Kan) after treatment with cefotaxime (0, 50, 100, 200 mg l^-1) (Cef) in combination with AgNO₃ to evaluate the frequency of plasmid loss. The presence of AgNO₃ was correlated with a decrease in plasmid stability in the presence of Cef (50 mg l^-1). Colonies that developed on non-Kan plates were analyzed by PCR to determine if functional copies of the nptII gene were still present after treatment with AgNO₃ and Cef. The effect on the growth and survival of bacterial cells investigated here may provide a useful clue to the interaction between plants and Agrobacterium during and after co-cultivation in the presence of AgNO₃, taking advantage that this chemical also stimulates plant regeneration.

Emilio Cervantes, Juana G. de Diego (Spain) Morphological Description of Plants: New Perspectives in Development and Evolution (pp 68-71)

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Opinion Paper: The morphological description of plants has been fundamental in the history of botany and provided the keys for taxonomy. Nevertheless, in biology, a discipline governed by the interest in function and based on reductionist approaches, the analysis of forms has been relegated to second place. Plants contain organs and structures that resemble geometrical forms. Plant ontogeny may be seen as a sequence of growth processes including periods of continuous growth with modification that stop at crucial points often represented by structures of remarkable similarity to geometrical figures. Instead of the tradition in developmental studies giving more importance to animal models, we propose that the modular type of plant development may serve to remark conceptual aspects in that may be useful in studies with animals and contribute to original views of evolution.