Volume 2 Number 1 2008

CONTENTS AND ABSTRACTS

K. Padmalatha, M.N.V. Prasad (India) In Vitro Plant Regeneration of Pterocarpus santalinus L.f (Red Sanders) – An Endangered Medicinal Plant and Important Timber Tree (pp 1-6)

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Original Research Paper: This paper describes multiple shoot regeneration of Pterocarpus santalinus L.f, an endangered tree endemic to the Deccan region which is commercially and medicinally most valuable for its heartwood on the international market. Seed germination under in vivo (in field conditions) and in vitro conditions were 60% and 100%, respectively. The nodal segments from in vitro regenerated shoots and from mature trees proliferated into multiple shoots (mean = 6.5) on Murashige and Skoog (MS) medium supplemented with 6-benzyl amino purine (BAP), kinetin (KN) and thidiazuron (3.0 mg L^-1) individually. The seed explants (embryonic axis along with the cotyledons), when cultured on MS medium containing a combination of KN and BAP (1.0 + 2.0 mg L^-1) formed 19-20 multiple shoots. MS basal medium was found suitable for rooting (70-80%). On transfer to a glasshouse 20% (3 plantlets/explant) survived.

Ravindra B. Malabadi (Canada/India), Jaime A. Teixeira da Silva (Japan), K. Nataraja (India) Stable and Consistent Agrobacterium-Mediated Genetic Transformation in Pinus roxburghi (Chir Pine) (pp 7-13)

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Original Research Paper: This paper highlights an Agrobacterium tumefaciens-mediated transformation protocol, developed for embryogenic cell cultures derived from vegetative shoot apices of mature, 14 years-old trees of Pinus roxburghii. The plasmid pBI121, containing the neomycin phosphotransferase II (nptII) gene providing kanamycin resistance as a selectable marker and the β-glucuronidase (uidA/GUS) reportergene, was used as a binary vector. Transformation frequencies were dependent on the species, genotype and post-cocultivation procedure. The highest transformation efficiency was obtained in the embryogenic line PR105 (37 transformed lines/g fresh wt) than with the embryogenic lines PR11 and PR521. The transgenic state of the embryogenic tissue was initially confirmed by histochemical GUS assay. Stable integration of the nptII gene in the plant genome of P. roxburghii was confirmed by polymerase chain reaction (PCR), Southern and Northern blot analyses. These results demonstrated that a stable and enhanced transformation system has been established in chir pine, and that this system would provide an opportunity to transfer economically important genes into other genotypes of P. roxburghii.

Yuping Ling, Li Li, Lili Wang, Bin Pan, Bo Wang, Peng Chen (China) Ultrastructural Study of Transcytosis in Parenchymal Tissues of Ginkgo biloba (pp 14-17)

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Original Research Paper: Transcytosis was examined in the parenchymal cells of the vegetative tissues of Ginkgo biloba with electronic microscopy, with the following results. (1) There are two possible modes of transcytosis based on morphological characteristics, involving either the transportation of pit fields or endocytosis and exocytosis. No plasmodesmata were observed in the parenchyma of any vegetative ginkgo tissue. (2) Pit fields in the cell walls of the parenchyma are simple symmetric pits, with 3–5 usually joined together like beads. The thickness of the adjacent cell walls is about 1 μm, and the diameter of the narrowest pit field is 0.2–0.3 μm. Cellular organelles congregate near the pit. (3) There is a great deal of endocytotic and exocytotic activity in the parenchymal cells. There are at least two forms of endocytosis: receptor-mediated endocytosis and fluid-phase endocytosis, in which ellipsoid or spherical microcysts with diameters of approximately 0.05–0.1 μm, separately imbibe the large macromolecular liquid-phase substances dissolved in the matrix. (4) When endocytosis and exocytosis begin, the part of the plasma membrane next to the cell wall sinks, forming a ligand-coated pit, afterwards wrapping the receptor within the caveola. The plasma membrane near the cell wall adheres to it and separates from the plasma membrane to form coated vesicles, which then enter the cell. Finally, the ligand partially or entirely decomposes, and the caveola enters the cell via a lysosome. (5) In mesophyll cells, the plasma membrane extends gemma-like extrusions after it sinks, part of which form sub-gemma. These separate from the venter-gemma to enter the cytosol. In endocytosis, there are two types of sub-gemma vesicles: the electron density of one is low, similar to that of the liquid phase, and the other has a rich fibrous stripe structure.

Ali Sarkhosh, Zabihollah Zamani, Reza Fatahi, Hamid Ranjbar, Mohammad Reza Vazifeshenas (Iran) Evaluation of Iranian Soft-seed Pomegranate Accessions by using Simple and Multivariate Analyses (pp 18-25)

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Original Research Paper: An important aspect of soft-seed pomegranates is their pleasant organoleptic character for breeding programs. In this study some quantitative and qualitative characters of 21 Iranian soft-seed pomegranate fruits and their seed components were recorded for categorizing the accessions. Analysis of variance showed that all of the characters in the examined accessions were significant, showing high variability. Results of bivariate simple correlation analysis showed the existence of significant positive and negative correlations among some important characters. Factor analysis showed that fruit length, fruit width and fruit juice, aril and seed characters composed the main factors. The most effective characters were categorized into 10 main factors (with an Eigen value ≥ 1) that contributed to 91.51% of total variance. For each factor, loading value of more than 0.5 was used as the significant threshold level. Cluster analysis was performed using these 10 factors and genotypes at a distance of 10 out of 25 were divided into 4 main clusters. These groups were mainly distinguished by their soft seededness, and aril and fruit size. Furthermore, by using three main factors, genotypes were plotted in 3 dimensions, in which accession Bihaste Shirin Khabre Baft with hard seeds was separated from the rest of the accessions.

Stephen J. Trueman, Donna M. Richardson (Australia) Relationships between Indole-3-Butyric Acid, Photoinhibition and Adventitious Rooting of Corymbia torelliana, C. citriodora and F₁ Hybrid Cuttings (pp 26-33)

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Original Research Paper: Rooted cuttings propagation is widely used for maximising tree yield, quality and uniformity in conjunction with clonal selection. Some eucalypt species are deployed as rooted cuttings but many eucalypts, such as the spotted gums (e.g. Corymbia citriodora), are considered too difficult-to-root to be produced as cuttings. This study examined IBA effects on photoinhibition, root formation, mortality, and root and shoot development of cuttings of C. torelliana, C. citriodora and their widely-planted F₁ hybrids. IBA had little or no effect on photoinhibition but it had strong, dose-dependent effects on root formation and mortality. IBA frequently increased primary root number of rooted cuttings but it did not increase total root weight, length, surface area or volume, possibly because the highest dose (8 g IBA/kg powder) caused leaf abscission and sometimes reduced leaf area (by 55–79%) or shoot dry weight (by 40–58%). An intermediate dose (3 g IBA/kg powder) most consistently improved root formation with little or no effect on mortality or shoot development. Across the F₁ hybrid families, this treatment increased the number of rooted cuttings by 72–121% and more than doubled the number of primary roots per rooted cutting (from 1.1–1.7 roots to 3.5–4.1 roots). This simple treatment will facilitate commercial multiplication of superior individuals or selected families of C. torelliana × C. citriodora through a vegetative propagation system.

Ravindra B. Malabadi (Canada/India), Jaime A. Teixeira da Silva (Japan), K. Nataraja (India) Salicylic Acid Induces Somatic Embryogenesis from Mature Trees of Pinus roxburghii (Chir pine) using TCL Technology (pp 34-39)

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Original Research Paper: Several physiological and biochemical effects of salicyclic acid (SA), when applied to plants, have been known for a long time. This paper highlights for the first time the role of SA as a signaling molecule in inducing embryogenic tissue derived from mature trees of Pinus roxburghii. External pre-treatment of explants with different concentrations (0.1, 0.2, 0.4, 0.5, 1.0, 2.0, 5.0 mg^-1) of SA for 5 min could not induce somatic embryogenesis effectively when plated on DCR induction medium. All explants of 10 genotypes failed to induce embryogenic tissue, and resulted in the browning of explants with callusing during pre-treatment of explants with SA. On the other hand, incorporation of 1.0 mg^-1 SA in full DCR medium, i.e. containing 2,4-D, NAA and BA, was optimum for all Chir pine genotypes by increasing the percentage of somatic embryogenesis significantly more than the control. The highest percentage (31%) of somatic embryogenesis was recorded in genotypes PR-821 and PR-46. SA proved to be a powerful synergistic activator of somatic embryogenesis in conifers when placed with other plant growth regulators.

Felix D. Ugese_, Paul K. Baiyeri, Benjamin N. Mbah (Nigeria) Mineral Content of the Pulp of Shea Butter Fruit (Vitellaria Paradoxa C. F. Gaertn.) Sourced from Seven Locations in the Savanna Ecology of Nigeria (pp 40-42)

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Short Communication: Fruit samples of the shea butter tree, Vitellaria paradoxa, were collected from seven locations across the southern Guinea savanna, northern Guinea savanna and Sudan savanna of Nigeria in the early part of the rainy season (the main period for fruit fall) in 2006. Oven dry samples of fruit pulps were subjected to laboratory analysis to determine their mineral nutrient content. Results of analysis of variance showed remarkable variations in Na, K, Mg and Fe content across the three ecologies. Similarly, all nutrient elements considered were significantly (P < 0.05) influenced by location (accession). Accession obtained from Lokoja recorded higher concentrations of Na, P and K, but the Kano accession had higher values for Ca, Mg, Fe and Zn. Principal component analysis appeared to suggest Na, P, Fe and Zn as nutrient traits capable of being used in classifying shea fruit pulp in the savanna of Nigeria. There were significant positive correlations among the nutrient elements. Relatively high values of nutrient elements as obtained in this study suggest that Shea butter fruit pulp is capable of combating malnutrition by meeting the nutritional requirement of rural dwellers among which shea fruit is already recognized and cherished as a food source.

Manuel Fernández, Reyes Alejano, Luis Domínguez, Raúl Tapias (Spain) Temperature Controls Cold Hardening more Effectively than Photoperiod in Four Mediterranean Broadleaf Evergreen Species (pp 43-49)

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Original Research Paper: Forestry plantations with evergreen broadleaf species in Mediterranean climate sites usually perform poorly in the field. Holm oak (Quercus ilex ssp. ballota (Desf.) Samp.), cork oak (Quercus suber L.), wild olive tree (Olea europaea L. ssp. europaea var. sylvestris) and lentisk tree (Pistacia lentiscus L.) are extensively used for such plantations. In order to determine the environmental factor that induces cold hardening most effectively and the mechanisms that are involved in this process, seven month-old nursery seedlings were taken to three growth chambers during the hardening phase and submitted to: 22/17ºC (day/night) and decreasing photoperiod from 12 to 8 h (Ph chamber), 12 h photoperiod and decreasing temperature from 22/17ºC to 8/3ºC (T chamber); and progressive reductions in temperature and photoperiod (PhT chamber). The variation of morpho-physiological traits was assessed. Reducing the photoperiod by up to 8 h did not stop the growth in height and diameter. Air temperatures below 8ºC reduced substantially height growth, mainly in Quercus species, but not diameter growth. Reducing the temperature proved much more effective for cold hardening than reducing the photoperiod. Low temperatures induced seedlings to accumulate non-structural carbohydrates (soluble sugars in the leaves and roots, and starch in the roots), and to improve cold hardiness. Differences among species were observed in cold hardiness, with Q. ilex > Q. suber ³ P. lentiscus ³ O. europaea. The maximum level of cold hardiness achieved in this experiment by Q. suber and Q. ilex was reached when seedlings accumulated 775-800 h₈ (hours ≤ 8°C), 750 h₈ in O. europaea and 725 h₈ in P. lentiscus. Finally, measuring chlorophyll fluorescence (Fv/Fm) after a freezing test was useful in estimating the cold hardiness of these species, providing results in less than two days for plant quality purposes.

Edison Paulo Chu, Armando Reis Tavares, Rosete Pescador, Kátia Crivelari Tonello, Erika Szeibel Yokota (Brazil) Callus Induction in Caesalpinia echinata, a Brazilian Endangered Tree (pp 50-53)

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Original Research Paper: Caesalpinia echinata (Brazilwood) is an endangered tree from Brazil. Its exploitation due to the extraction of its red dye (brazilin) is intimately connected with the history and colonization of Brazil. Tissue culture can be used as a potential method for vegetative multiplication, which can provide a high rate of selected plants. Leaf fragments, stem segments and seed primordia from small and large fruits were used as explants, and different balances of auxins (IAA, 2,4-D and NAA) and cytokinins (BA, zeatin, kinetin and thidiazuron) were used to induce organogenesis. Despite the various auxins, auxin/cytokinin balances and reduction in MS salts, only induction of callus was observed in the modified MS medium without any differentiation, with progressive oxidation after 40 days of experimentation. Seed primordia from small fruits formed a whitish callus, and the best results expressed as percentage of induced callus corresponded to 0.5 mg L^-1 TDZ and 4 mg L^-1 2,4-D treatments. Microscopic analysis of the callus showed elongated cells, inadequate for multiplication and dedifferentiation as well as round cells with a large nucleus, appropriate to cell division and organogenesis. Callus induction occurred in most treatments with growth regulators, but the species showed recalcitrance to differentiation, an unsuitable characteristic for organogenesis.

Tamilmani Eevera, Kaliyaperumal Rajandran, Saravanan Saradha, Amirthalingam Lashmi (India) Analysis of Genetic Variation in Selected Bamboo Species using RAPD (pp 54-56)

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Research Note: Taxonomic and systematic studies of bamboos are traditionally based on floral morphology, which can cause problems in identification due to the lack of or infrequent flowering. There are limited studies using molecular techniques to overcome taxonomic problems in woody bamboos. In this study RAPD was used to differentiate 26 bamboo species. Screening was done with 50 random primers and consistent results were obtained with about 10. Dendrocalamus species stood out among other bamboo species.

Rosilaine Carrenho, Fabio F. Barbosa, Cristiano V. M. Araújo, Lander J. Alves, Osvaldo M. Santos (Brazil) Mycorrhizal Associations in Eucalyptus spp.: Status and Needs (pp 57-67)

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Review: In nature, there are some intra- or interspecific ecological relations with different dependence levels. Plant roots are good examples of this, because they develop ecological relations with many soil microorganisms, mainly with the fungi, forming mycorrhizae. The interest in studying this association has risen up due to increasing necessity of alternative handling methods that promote improvement in the plant growth and productivity. Arbuscular mycorrhiza have been known and explored for a long time due to the ecological significance and effects in the growth and nutrition of plants, playing an important role for the nutrient uptake system. The mycorrhizal establishment in eucalyptus has been known for many years, and the benefits of this symbiosis have been commercially explored. The most recent outcomes regarding the occurrence, importance and use of arbuscular mycorrhiza in Eucalyptus spp. are presented in this paper. Firstly, these will be grouped in various research topics (state of the art, mycorrhizal dependence; colonization and benefits; diversity of arbuscular mycorrhizal fungi in eucalyptus forests; coexistence of arbuscular-and ecto-mycorrhizal fungi; perspectives and needs). Then, a brief discussion will take place in order to define the real state of knowledge of this subject related to Eucalyptus spp.