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Functional Plant Science and Biotechnology

Volume 1 Number 1 2007

FPSB


CONTENTS AND ABSTRACTS

P. Venkatachalam (USA/India), P. Kumari Jayasree, S. Sushmakumari, R. Jayashree, K. Rekha, S. Sobha, P. Priya, R.G. Kala, A. Thulaseedharan (India) Current Perspectives on Application of Biotechnology to Assist the Genetic Improvement of Rubber Tree (Hevea brasiliensis Muell. Arg.): An Overview (pp 1-17)

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Special Feature: Hevea rubber clones are intensively cultivated in tropical regions of the world as main sources for Natural Rubber (NR) production which is one of the important raw materials for many industries. However, the heterozygous nature, long breeding time of trees and the prolonged period required for evaluation of mature traits are strong limitations for conventional breeding and selection methods. The development of methods for in vitro culture and genetic engineering has increased the possibility of producing rubber genotypes with improved latex yield, tolerance to tapping panel dryness (TPD) syndrome, growth rate and wood quality or reduction in undesirable traits. The combination of conventional breeding and molecular techniques will help to develop rubber trees with positive effects on the environment. However, the risks associated with the biotechnological applications should be carefully evaluated and field trials are to be performed with transgenic rubber tree. Genomic technologies were taken up by various research groups working on Hevea to identify new targets for breeding and/or complementary genetic transformation. In addition, molecular markers can provide simultaneous and sequential selection of agronomically important genes in Hevea breeding programs and effectively replace time consuming bioassays in early generation screens. With the advent of molecular techniques, several genes involved in rubber biosynthetic pathway have been characterized. Both HMGR and HMGS are essential enzymes involved in early steps of rubber biosynthesis. Among the genes identified, REF is a key rubber biosynthetic gene involved in polymerization of isoprene chain. However, further research is needed to use these identified genes for genetic manipulation of rubber tree. Most recently, a set of genes associated with TPD has been identified by SSH analysis. This review provides a comprehensive picture on rubber biotechnological research achievements in the last two decades.

Miguel A. Duarte-Vázquez, Sandra García-Padilla, Blanca E. García-Almendárez, Carlos Regalado (Mexico) Purification of Natural Plant Peroxidases and their Physiological Roles (pp 18-31)

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Invited Review: The plant peroxidase superfamily comprises heme-containing glycoproteins that differ in their structure and catalytic properties. POX has been isolated and characterized from a large number of plant sources like fruits, leaves, tubers and grains, and the major source of commercially available peroxidase is horseradish roots. However, availability of POX with higher stability and different specificity would improve immunoenzymatic analytical kits and promote the development of new analytical methods and potential industrial processes. Therefore, extensive investigations of several peroxidases of different origins have been reported. The diversity of reactions catalyzed by plant peroxidases (POX) accounts for the implication of several isoenzymes in a broad range of physiological processes including indole-3-acetic acid metabolism, pathogen resistance, response to oxidative and chemical stresses involving reactive oxygen species, and lignin and suberin biosynthesis. In addition, the recent description of the hydroxylic cycle which leads to the formation of various radical species, opens a new range of implications for these enzymes. A major limitation for the widespread use of POX is the current high cost of production of the enzyme. A cost-effective purification technology and alternative sources with high peroxidase activity can help bring down the cost of this enzyme production.

Rüdiger Hardeland (Germany), S.R. Pandi-Perumal (USA), Burkhard Poeggeler (Germany) Melatonin in Plants - Focus on a Vertebrate Night Hormone with Cytoprotective Properties (pp 32-45)

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Invited Review: Melatonin, first described as a vertebrate hormone produced by the pineal gland, is, in fact, a ubiquitous substance found in the majority of taxa studied, from bacteria and eukaryotic unicells to fungi, pheophyceans, rhodophyceans, chlorophyceans and angiosperms. Since one of the first physiological functions discovered was that of mediation of the signal gdarknessh, several studies in photoautotrophs were also focussed on this aspect, with particular attention to photoperiodism. In some species, e.g., the dinoflagellate Lingulodinium and the dicot Chenopodium, melatonin exhibits a robust circadian rhythm peaking at night. The precise role of this rhythmicity is not fully understood and may serve functions other than photoperiodic control. Circadian variations are not always detected or can be differently phased. They should be absent in dry oily seeds, which are particularly rich in melatonin. High quantities are also reported for several medicinal plants and plants exposed to intense natural UV radiation. Not all reports on melatonin levels in photoautotrophs are equally reliable, for methodological reasons. Nevertheless, high amounts found in a number of well-designed studies, also in relation to UV, indicate that cytoprotective properties known from animals may play a role in plants, too. In dry seeds, free radicals cannot be detoxified enzymatically. Therefore, melatoninfs scavenging properties could be of biological value and contribute to antioxidative protection. Elevated levels in some juicy fruits may indicate a role during fruit ripening and a function in maintenance of developmental stages that may extend to the persistence of dormancy.

Han-Yi Fu, Shiang-Jiuun Chen, Ruei-Feng Chen, Ling-Long Kuo-Huang, Rong-Nan Huang (Taiwan) Why do Nettles Sting? About Stinging Hairs Looking Simple but Acting Complex (pp 46-55)

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Invited Review: To cope with environmental stress, plants have developed various defensive mechanisms against prey. For this purpose, some plants use external structures, like mechanical thorns and toxic stinging hairs. Although four families (Urticaceae, Euphorbiaceae, Loasaceae, Hydrophyllaceae) have genera including stinging hairs, most studies on stinging hairs are focus on nettles (Urtica spp.) in the Urticaceae. A stinging hair consists of one stinging cell and surrounding pedestal cells. A number of chemicals have been proposed as the toxins that are introduced through nettle stings when in contact with human skin, such as acetylcholine, histamine and serotonin, with formic acid being the most common nettle toxin. The nettle sting might induce significant pain, stinging and wheal reactions that may last >12 hours. Recent studies supporting oxalic acid and tartaric acid as persistent pain-inducing toxins in nettle stings suggest that the toxins in the stinging hairs may be fairly complex. The mechanism of the stinging reaction is still far from being understood. This review summarizes some previous studies with additional assumptions in order to suggest some theories regarding synthesis, storage, and secretion of sting toxins. The immunological and some other physical responses after the skin is stung are also discussed. Although much of the knowledge from the stinging reaction is still fragmented and mysterious, the tiny structures and mechanisms of the stinging hairs are still amazingly interesting.

Simcha Lev-Yadun, Nitsa Mirsky (Israel) False Satiation: The Probable Antiherbivory Strategy of Hoodia gordonii (pp 56-57)

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Research Note: The succulent, thorny, South African desert plant Hoodia gordonii and several other species of the genus, as well as Pectinaria maughamii were found to be appetite suppressants and thirst quenchers. The appetite-suppressant drug from Hoodia species increases the content of ATP by 50-150% in hypothalamic neurons, thus activating an anorectic mechanism. We propose that these plants have evolved such compounds as a defense against herbivory, causing false satiation. We hypothesize that the same principle operates in other plants, such as Thaumatococcus daniellii that produces nature's sweetest known molecule (the protein thaumatin). Hoodia and similar plants thus express a previously unknown mechanism of anti-herbivore defense - false satiation.

Cástor Guisande, Carlos Granado-Lorencio (Spain), Camilo Andrade-Sossa, Santiago Roberto Duque (Colombia) Bladderworts (pp 58-68)

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Invited Review: The aim of this review is to give a general idea of the current knowledge of the habitat, phylogeny, biogeography, reproduction, and carnivory of the genus Utricularia. These carnivorous species can be grouped according to into terrestrial, free-aquatic, affixed-aquatic, rheophytic, lithophytic and epiphytic habits, although many species have the capacity of inhabiting several different habitats. The terrestrial habit has been phylogenetically optimised as the ancestral condition, with the epiphytic and aquatic habits found to be mostly derived. The genus is cosmopolitan and phylogenetic optimizations of biogeographic area indicate a probable Neotropical origin. Some Utricularia species display almost complete sterility, and vegetative reproduction via turions, tubers and shoot fragments plays a dominant role in the establishment of these populations. There is controversy about the importance of insect pollination. Utricularia species do not face the pollinator-prey conflict, and the large flowered epiphytic species are probably derived, therefore, it seems likely that insect pollination may play an important role in the sexual reproduction of some Utricularia species. Bladders (utricles or traps) capture and digest prey, however strategies of chemo-attraction have not been determined. Finally, the benefit of carnivory is discussed in terms of the costs to photosynthetic rate and respiration associated with the production and maintenance of bladders.

Sally L. Hanton, Loren A. Matheson, Laurent Chatre (Canada), Federica Brandizzi (Canada/USA) The Golgi Apparatus - A Key Organelle in the Plant Secretory Pathway (pp 69-76)

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Invited Mini-Review: The secretory pathway in plants mediates the transport of cargo molecules from their site of synthesis to other compartments within the pathway as well as to the cell wall. At the centre of this system is the Golgi apparatus, a transport hub that is responsible for receiving, modifying and sorting proteins for transport to their destinations. Several recent publications have investigated the routes and transport mechanisms that connect the Golgi apparatus with other secretory organelles, including exciting new links with non-secretory organelles such as plastids. This review therefore focuses on the multiple roles of the Golgi apparatus in the secretory pathway and its connections with other organelles within the plant cell.

Trinh Xuan Hoat, Kouhei Uchihashi, Hitoshi Nakayashiki, Yukio Tosa, Shigeyuki Mayama (Japan) Programmed Macromolecule Degradation during Apoptotic-Cell Death in Oats (pp 77-84)

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Invited Mini-Review: The execution of programmed cell death (PCD) involves the controlled degradation of cellular macromolecules such as proteins and nucleic acids. Compared with animal systems, very little is known about the molecular mechanisms regulating macromolecule degradation during plant PCD. Victorin, a host-selective toxin produced by the fungus Cochliobolus victoriae, induces PCD in oat cultivars harboring the Vb gene. Victorin-induced PCD displays typical morphological and biochemical features of apoptosis, including nuclear DNA laddering, chromatin condensation, cell shrinkage, and a mitochondrial permeability transition. In the oat-victorin system, it has been demonstrated for the first time that certain cellular macromolecules are specifically degraded during plant PCD. One example is the specific proteolytic cleavage of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Two subtilisin-like serine proteases that exhibit caspase-like activity have been identified as associated with Rubisco proteolysis. Another example involves the degradation of RNA molecules. Ribosomal RNA species from the cytosol, mitochondria and chloroplasts are all degraded via specific degradation intermediates during victorin-induced PCD. Concurrently with rRNA degradation, mRNAs of housekeeping genes such as actin and ubiquitin but, interestingly, not those of stress-responding genes such as PR-1 and PR-10, are also targeted for specific degradation. The oat-victorin system, therefore, serves as a model for elucidating the molecular mechanisms regulating macromolecule degradation in the execution phase of plant PCD.

Angel J. Matilla (Spain) How is the Silique Fruit Dismantled over its Maturation? (pp 85-93)

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Invited Review: In dehiscent fruits, such as the silique of Arabidopsis, housed ripe seeds are dispersed into the surrounding environment through a process known as pod shattering. This seed-expelling process is a consequence of the partial and gradual dismantling of the silique architecture. The shattering occurs at a precise site in the silique (i.e. valve margins, made up of a separation layer and adjacent lignified layer) and involves a network of tightly regulated genes. Thus, (i) INDEHISCENT (IND) primarily directs the differentiation of the valve-margin cells into the separation and lignified layers; (ii) SHATTERPROOF (SHP1, SHP2), ALCATRAZ (ALC ) and IND directs the valve-margin identity and pod shattering; (iii) SHP, ALC, IND and FRUITFULL (FUL) are required for lignification of the most internal valve-cell layer (enb); (iv) REPLUMLESS (RPL) and FUL have been found to set the boundaries of the genes that confer valve-margin identity; (v) FUL acts primarily in the valve to restrict the expression of IND, SHP, and ALC to the valve margin, rather than by playing a major role itself in specifying valve identity; (vi) RPL maintains the replum boundary by restricting the expression of SHP to the valve margin; (vii) JAGGED (JAG), that promotes lateral organ growth, and YABBY3 (YAB3) and FILAMENTOUS FLOWER (FIL), which are both related to establishing abaxial polarity in lateral organs, are necessary for expression of FUL and SHP in the valve and valve margin, respectively; and (viii) RPL regulates SHP indirectly by restricting JAG and FIL expression from the replum. In this review, knowledge concerning the opening of Arabidopsis fruit is compared with other still less-known crucifer and non-crucifer species.

Nicholas Vrettos (Greece), Barthélémy Tournier (Canada) The Sound of Silence in Plants (pp 94-99)

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Invited Mini-Review: RNA silencing appears as one of the most exciting discoveries in the field of molecular biology of the last decade. Double-stranded small RNA species ranging from 20 to 26 nt, named siRNAs, were initially identified as a plant defense mechanism to target viral RNA upon infection. siRNAs can bind to a complementary RNA and induce its degradation. In addition, single stranded miRNAs have been characterized as endogenous small non-coding RNAs involved in mRNA inhibition of translation or cleavage in both plants and animals. A broad range of physiological processes are related to this class of RNAs such as control of development, defense against viral infection and cancer. RNA silencing involves several RNA processing enzymes which are well conserved between plants and animals. However plants have developed specific features of RNA silencing that will be reviewed here. Plants and C. elegans are the only organisms capable of producing double stranded RNA from single stranded molecules when these possess specific qualitative and quantitative features. RNA dependent RNA polymerases (RDR) are the critical enzymes for this step. A special focus will be made in this review on the recent progress in characterizing the ability of plants to send short and long distance signals from the initially silenced cell.

Adolfo Rosati (Italy) Physiological Effects of Kaolin Particle Film Technology: a Review (pp 100-105)

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Invited Mini-Review: Particle film applications (i.e. spraying canopies with a suspension of particles of various kinds of clay, including kaolin, leaving a film on the leaves) has long been used to limit the impact of water and heat stress on crops. Earlier work has focused primarily on crop yield and suggests that particle film applications, in some crops and under some conditions, increases yield. More recent and detailed work has been carried out using new kaolin products. Such work suggests that, besides other effects on fruit colour and size, kaolin generally reduces photosynthetic rates of individual leaves except under high temperature and/or heat stress. This is probably because kaolin films increase the albedo thus reducing leaf temperature and the consequent heat stress, but also reducing the light available for photosynthesis, possibly offsetting benefits of lower temperature, depending on the level of heat stress and incident irradiance. The few studies on the effects of kaolin applications on canopy photosynthesis report either an increase, or no effect, despite a reduction in photosynthetic rates of individual leaves. This is probably due to improved light distribution within the canopy, which increases the radiation use efficiency more than compensating for the slight reduction in canopy light absorption. In conclusion, kaolin applications appear to have the ability to reduce the effects of water and/or heat stress and, possibly to enhance canopy photosynthesis, at least under certain circumstances. These effects alone might not necessarily justify kaolin applications economically. However, when the kaolin film technology is adopted for pest management or for other purposes, it can be concluded that there are possible additional benefits.

Paul Hein, Ralf Oelmüller (Germany) Photosystem I and Regulatory Proteins for its Biogenesis (pp 106-111)

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Invited Mini-Review: Photosystem I (PSI) is a pigment-protein complex located in the thylakoid membrane of cyanobacteria and chloroplasts of algae and higher plants, which functions as a plastocyanin (or cytochrome c6)-ferredoxin oxidoreductase. The reducing potential of ferredoxin is utilized for a variety of biochemical processes such as the reduction of NADP+, or the assimilation of nitrate or sulfate. In higher plants the complex consits of 14 different polypeptide subunits, nine of them are encoded in the nucleus (PSI-D/E/F/G/H/K/L/N/O) and the residual five (PSI-A/B/C/I/J) are plastid-encoded. Most of the information about the function of PSI derives from mutants impared in one or more subunit genes or from biochemical studies. In 2003, the crystal structure of PSI from a higher plant was determined. In contrast to the trimeric cyanobacterial PSI, the plant PSI was purified as a monomer. Compared to the structural information, much less is known about the regulation and assembly of PSI and only a few regulatory factors have been identified so far. In this review we describe the structure of PSI and the role of the individual subunits and present an overview on those factors which are required for PSI acumulation in pro- and eukaryotic photosynthetic organisms.

Georgios Liakopoulos, Dimosthenis Nikolopoulos, George Karabourniotis (Greece) The First Step from Light to Wine: Photosynthetic Performance and Photoprotection of Grapevine (Vitis vinifera L.) Leaves (pp 112-119)

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Invited Review: Grapevine (Vitis vinifera) cultivation is successfully established across large areas of the globe whose environmental conditions range from those of the northern temperate zone of the Rhine valley to those of the driest and hottest regions of the Greek islands and the Californian desert. The photosynthetic metabolism follows the C3 pathway and the responses of the photosynthetic machinery to essential environmental factors are similar to other C3 plants. The high adaptability of vines to a plethora of different environments is derived from several photosynthetic and associated anatomical and biochemical characteristics of the grapevine leaf. The tight stomatal control coupled with the heterobaric construction of the leaf may offer advantages in water conservation and light utilization and therefore in the photosynthetic performance. Leaves of some cultivars may transiently be anthocyanic or pubescent, characteristics that are believed to be related to the protection against strong visible and ultraviolet radiation, especially during the sensitive stages of initial leaf development. An array of biochemical mechanisms renders the leaves of grapevine highly resistant to photoinactivation and lessens the possibility of photooxidative damage. Since grapevine leaves are the source of the biomolecules which determine the quantity and quality of the fruits, any knowledge on the leaf structure-function relationships and on the leaf-environment interactions is of particular importance to viticulture.

Yingjun Wang, Martin H. Spalding (USA) CO2 Concentrating Mechanisms in Eukaryotic Microalgae (pp 120-128)

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Invited Review: Many aquatic photosynthetic microorganisms possess inducible CO2 concentrating mechanisms (CCMs) that allow them to optimize carbon acquisition in environments with frequently changing and often limiting CO2 concentrations. The CCMs function by accumulation of a large quantity of intracellular inorganic carbon (Ci) through concerted Ci uptake systems and enzymes catalyzing the interconversion between different species of Ci. In addition, an array of regulatory devices appears present to facilitate the sensing of CO2 availability and the regulation of metabolic pathways. Over the past several decades, significant advances have been made in understanding the CCM and its regulation. With the aid of mutant studies and the availability of several cyanobacterial and eukaryotic algal genomes, an integrated picture is emerging to reveal many of the molecular details in the microalgal CCMs. This review will focus on the recent advances in identifying and characterizing the major components involved in the CCM, including Ci uptake systems and regulatory pathways in eukaryotic microalgae, especially in the model organism, Chlamydomonas reinhardtii.

Toshiharu Shikanai (Japan) The NAD(P)H Dehydrogenase Complex in Photosynthetic Organisms: Subunit Composition and Physiological Function (pp 129-137)

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Invited Review: The chloroplast NAD(P)H dehydrogenase (NDH) complex is believed to originate from the cyanobacterial NDH-1 complex because of their similarity in structure and function. In both cyanobacteria and chloroplasts, NDH is involved in respiratory electron transport (chlororespiration in chloroplasts) and photosystem I cyclic electron transport. By modifying its subunit composition, cyanobacterial NDH also contributes to CO2 concentration. In Arabidopsis thaliana, photosystem I cyclic electron transport consists of two partly redundant pathways. Although the knockout of chloroplast NDH gives only a subtle phenotype, it causes a severe defect in photosynthesis under the mutant background of pgr5 (proton gradient regulation 5), in which another pathway of photosystem I cyclic electron transport is impaired. Although the characteristics of the mutant phenotype suggest that PGR5-dependent photosystem I cyclic electron transport contributes markedly to ATP synthesis during photosynthesis, chloroplast NDH may function as a safety valve to prevent the over-reduction of stroma. In cyanobacterial and chloroplast NDH, we have not discovered the electron donor-binding subunits that are conserved in bacterial NDH-1. This fact implies that the NDH complex is equipped with a different electron-donor binding module in cyanobacteria and chloroplasts, and thus the identity of the electron donor to the complex is still unclear.

Miko U. F. Kirschbaum (New Zealand/Australia), Ülo Niinemets (Estonia), Dan Bruhn (Denmark), Anthony J. Winters (Australia) How Important Is Aerobic Methane Release by Plants? (pp 138-145)

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Invited Mini-Review: The first research paper describing aerobic methane release from living plants and dead organic matter was published in early 2006. These original findings have yet to be independently repeated and confirmed. Instead, the only other detailed study that has been published did not find any significant aerobic emissions of methane. Concerns remain about possible artefacts, especially with respect to methane adsorption and desorption. Several questions are yet to be answered, such as identification of a plausible biochemical mechanism for the process, how CH4 emissions might change with light, temperature or the physiological state of leaves, whether emissions change over time under constant conditions, whether they are related to photosynthesis and how they relate to the chemical composition of biomass. Various studies have assessed the likely magnitude of aerobic methane release within a global context. Different estimates based on more or less sophisticated approaches have all indicated that the magnitude of aerobic methane release must be relatively moderate and contribute between 0-10% of modern and 0-30% of pre-industrial/pre-agricultural methane emissions. In the context of land-use change, consideration of aerobic CH4 emissions from different plant types is only a small factor for overall greenhouse gas balances. Any carbon-offset benefit from planting trees is likely to be about 100 times as effective as any possible detrimental effect due to increased aerobic methane release. Land-use change, including the draining of wetlands, the establishment of paddy rice farming, or the introduction of ruminant animals, would produce emission changes that significantly outweigh any potential changes arising from differences in aerobic methane release by different plant types.

Roxana Zorzoli, Guillermo Raúl Pratta, Gustavo Rubén Rodríguez, Liliana Amelia Picardi (Argentina) Advances in Biotechnology: Tomato as a Plant Model System (pp 146-159)

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Invited Review: This review reports some aspects of advances in biotechnology such as tissue culture, genetic engineering, molecular markers, and the new approach of the gomicsh in tomato. In vitro regeneration has been a subject of research because of the commercial value of the crop and its amenability for further improvement via genetic manipulation. Numerous studies on plant regeneration from a wide range of tissues of wild and cultivated tomato germplasm have been conducted. Several genes involved in fruit ripening and others traits have been characterized and genetically engineered plants were successful obtained. One of the main uses of molecular markers has been the construction of linkage maps. Linkage maps have been utilized to identify chromosomal regions that contained genes controlling simple trait and QTL. DNA markers that are tightly linked to important genes are used as molecular tools for marker-assisted-selection (MAS) in tomato breeding. The latest trend is to combine QTL mapping with methods in functional genomics. There are wide collections of ESTs. The DNA microarrays analysis is also used to study the expression of many genes. Nowadays the proteomics and metabolomics allow identifying biochemical factors underlying important traits for tomato breeding programs.

M. Sujatha (India) Advances in Safflower Biotechnology (pp 160-170)

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Invited Review: Safflower is a versatile crop with several desirable attributes and multiple uses. Genetic improvement mostly relies on the variability available in the cultivar germplasm. Introgression of desirable traits from wild species is hampered by differences in basic chromosome number, asympatry and strong barriers to sexual crossability thus, necessitating the use of biotechnological tools. Reliable and reproducible protocols for direct as well as callus-mediated shoot regeneration from both somatic and gametic tissues through organogenic and embryogenic pathways are established. Compared to other Asteraceae members, the development of molecular and genomic resources for safflower has been limited. AFLP, ISSR and RAPD markers are used in assessment of genetic diversity in land races and germplasm and also the extent of outcrossing of cultivated safflower with weedy relatives. Noteworthy progress is being made in the development of a wide array of genetic engineering technologies for recombinant protein production using an oleosin-fusion protein system and metabolic engineering of oilseeds for production of high value lipids like -linolenic acid (GLA), eicosopentaenoic acid (EPA) and docosahexaenoic acids (DHA) for increased health and economic benefits. Owing to the self-pollinated nature of the crop, lower production and capital costs, greater production flexibility, safflower is used as a target for biopharming for production of plant made pharmaceuticals, novel proteins and industrial enzymes. The immediate challenge is to genetically engineer safflower for input (agronomic) traits and also for modification of safflower oil to high-value oil for specific market needs.

Nieves Villalobos, Luisa Martín, Antonio Blázquez, Juan Pedro Martín, Piedad Gallego, Beatriz Pintos, Hilario Guerra (Spain) Morphogenic Differentiation in Medicago (pp 171-194)

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Invited Review: Using somatic embryogenesis in Medicago arborea L. subsp. arborea and organogenesis in Medicago strasseri, we have achieved important advances in plant regeneration. The best results were obtained from M. arborea using petioles and anthers in the second phase of meiosis as explants. With M. strasseri, the best inducing explants were the petiole and leaf. The most efficient media were Murashige-Skoog (MS) medium with 2,4 dichlorophenoxyacetic acid (2,4-D) and kinetin. An attempt was made to optimise somatic embryogenesis by varying the composition of the culture medium. Embryogenesis was improved using proline but the collection of vigorous plants was poor. All (100%) of the somatic embryos obtained from anthers germinated and produced 63% green and 27% albino seedlings. The analysis of cytokinins in calli revealed clear differences in embryogenesis. The amount of total endogenous cytokinins: Z, [9R]Z, (diH)Z, (diH)[9R]Z, iP and [9R] iP, was greater in the non embryogenic than in embryogenic calli. The ratio between isopentyl derivatives and zeatin-like derivatives was lower in embryogenic than in non-embryogenic calli. Analysis of carbohydrates revealed that there were no significant differences in total sugars between embryogenic and non-embryogenic calli. The highest levels of reducing sugars were seen in embryogenic calli. During the development of somatic embryogenesis and organogenesis, very low starch levels were found in the calli. In embryogenic calli, the sucrose content was lower. Nitrogen levels decreased in the calli with an increase in culture time, mainly in embryogenic calli. Plant acclimatization was carried out over two months in glass pots containing sterile vermiculite and the plants were nourished every week with MS medium with half the concentration of salts. Then, they were transferred to sterile sand and after two weeks they were transplanted into flower pots. The humidity was gradually reduced over time. Of the somatic embryos induced, 28 } 3% developed into plants with normal phenotypic characteristics.

N.L. Raju, M.N.V. Prasad (India) Genetic Diversity Analysis of Celastrus paniculatus Willd. - a Nearly Threatened, Cognitive and Intelligence Enhancer - by RAPD Markers (pp 195-199)

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Original Research Paper: Celastrus paniculatus (Celastraceae) is a Red listed medicinal plant and the species has been overexploited over the last few decades for its medicinal uses. Information on its genetic diversity is currently lacking for strengthening its conservation management practices. Random amplified polymorphic DNA (RAPD) polymorphism was applied to check the efficiency of ex situ genetic conservation. Using this RAPD analysis, we observed a significant genetic variation among the collected accessions of C. paniculatus. Modified CTAB protocol yields a high quality DNA and the annealing temperature was standardized at 37C for RAPDPCR reactions. Fourteen random primers, each with ten base pairs generated 143 bands with 91% being polymorphic, which were used to estimate genetic distances among the collected accessions. The species show higher genetic diversity between accessions than other red listed medicinal plants. Mean locus similarity among individuals was 0.721 for all pairwise comparisons. The present work provides important baseline data for conservation and collection strategies for this species. Thus RAPD markers proved to be useful in distinguish variation in very rare species which are of conservation concern.

M. Thiruvengadam, K.T. Rekha, C.H. Yang (Taiwan, ROC) Somatic Embryogenesis and Plant Regeneration from Petiole-Derived Callus of Spine Gourd (Momordica dioica Roxb. ex Willd) (pp 200-206)

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Original Research Paper: An efficient protocol for plantlet regeneration from the cell suspension cultures of spine gourd (Momordica dioica Roxb. ex Willd.) through somatic embryogenesis is reported. Petiole-derived embryogenic calli were cultured on Murashige and Skoog medium augmented with 4.5 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 10% coconut milk (CM). The maximum frequency of somatic embryos (36.3%) was observed on MS medium supplemented with 2.2 M 2,4-D for three weeks of culture. Ontogenic studies of somatic embryos revealed that the cells destined to become somatic embryos divided into spherical proembryos and then progressed to globular, heart and further differentiated properly into torpedo and cotyledonary stages within 5 weeks. Embryo development was asynchronous and strongly influenced by the 2,4-D concentration. The MS liquid medium augmented with 2.2 M 2,4-D and 0.5 M L-glutamine was effective to achieve high frequency of somatic embryo induction (44.5%). The cotyledonary-stage somatic embryos were transferred to MS liquid medium with no plant growth regulators to achieve complete maturation within 7-days. Lack of 2,4-D in suspensions increased somatic embryo maturation with decreased abnormalities. Sucrose was found to be the best carbon source for callus induction, embryo maturation and embryo germination. Relatively, only few numbers of embryos developed into root/shoot when transferred to 1/10 MS solid medium containing 0.5 M abscisic acid (ABA), 2% (w/v) sucrose and 0.2% (w/v) Gelrite. About 11% of somatic embryos germinated into morphologically normal fertile plants within 2 weeks. Regenerated plantlets were successfully hardened, with a survival rate of approximately 60%, and established in the field. This regeneration protocol assured successful embryo induction, maturation and plantlet conversion.

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