tag:blogger.com,1999:blog-83938970179908918502023-11-15T09:01:47.775-08:00info4youUnknownnoreply@blogger.comBlogger4125tag:blogger.com,1999:blog-8393897017990891850.post-6569054735910534272010-08-31T04:23:00.000-07:002010-08-31T04:44:38.803-07:00Orthodox and Recalcitrant SeedsCHAPTER 4<br /><br /><br />Orthodox and<br />Recalcitrant Seeds<br /><br /><br /><br /><br /><br />P A T R I C I A B E R J A K and<br />N.W. PAMMENTER<br />Plant Cell Biology Research Unit,<br />School of Life Sciences<br />University of Natal, Durban<br />4041 South Africa<br /> <br /><br /><br /><br /><br />The seeds of many species cannot be classified as ortho-<br />dox, and this is particularly so in the case of tropical tree seeds.<br />The views presented here favor a continuum of seed behavior<br />that is based on a variety of characteristics. A suite of mecha-<br />nisms or processes is discussed that embodies the properties<br />currently thought to promote the acquisition of desiccation<br />tolerance and to ensure survival of the desiccated condition in<br />orthodox seeds. These include: cellular and intracellular phys-<br />ical characteristics; intracellular de-differentiation; the<br />“switching off” of metabolism; the presence and efficient<br />operation of antioxidant systems; the accumulation and roles<br />of putatively protective molecules, including late embryogenic<br />accumulating/abundant proteins (LEA’s), sucrose, and certain<br />oligosaccharides; deployment of amphipathic molecules; an<br />effective peripheral oleosin layer around lipid bodies; the<br />occurrence and operation of repair mechanisms during rehy-<br />dration; and others yet to be identified. The presence of some<br />of the mechanisms/processes, or their absence or partial<br />expression, is considered in the context of the varied respons-<br />es to dehydration shown by nonorthodox seeds. The factors<br />that determine distinct variations in the behavior of recalci-<br />trant seeds of individual species under the same conditions is<br />given attention, with the effects of drying rate (i.e. the rate of<br />water loss from tissues of desiccation-sensitive seeds) being<br />stressed. Two different factors are distinguished in this regard:<br /> <br /><br /><br /><br /><br /><br />(1) damage that occurs at low water contents when nonfreez-<br />able water, which is held to stabilize intracellular structures<br />and macromolecules, is removed, which is desiccation damage<br />in the strict sense; and (2) damage that occurs during slow<br />dehydration, when metabolic imbalances are proposed to<br />cause the generation of damaging chemical species, e.g. free<br />radicals, which is termed metabolic damage. Desiccation dam-<br />age, in the strict sense, is attributed to the lack or inadequate<br />operation of the processes/mechanisms held to protect desic-<br />cation-tolerant seeds in the dry state, while metabolic damage<br />is considered in the context that nonorthodox seeds (especial-<br />ly those that are truly recalcitrant) do not possess the suite (or<br />full suite) of mechanisms/processes that facilitate the acquisi-<br />tion and maintenance of desiccation-tolerance as exhibited by<br />maturing and mature orthodox seed-types.<br /><br /><br /><br /><br />INTRODUCTION<br /><br /><br /><br />Orthodox seeds (Roberts 1973) acquire desiccation tolerance<br />during development and may be stored in the dry state for pre-<br />dictable periods under defined conditions. Unless debilitated<br />by zero-tolerant storage fungi, orthodox seeds should maintain<br /><br /><br />Chapter 4: Orthodox and Recalcitrant Seeds <br /><br /><br /><br /><br />high vigor and viability at least from harvest until the next<br />growing season (Berjak and others 1989) or for many decades<br />at -18 °C (IBPGR 1976). Generally, such seeds undergo a peri-<br />od of drying during their maturation and are shed at low water<br />content which is in equilibrium with the prevailing relative<br />humidity (r.h.). The equilibrium water content at any particu-<br />lar r.h. is determined by seed composition, but all orthodox<br />seeds can withstand dehydration to around 5 percent (0.053 g<br />H2O g-1 dry material [g g-1 ]), even when maturation drying is<br />not completed prior to shedding. Any seed that does not<br />behave this way is not orthodox, and, in fact, the seeds of a<br />great number of tropical species may accordingly be nonortho-<br />dox. Nonorthodox seeds have so far been described as either<br />being recalcitrant (Roberts 1973) or intermediate (Ellis and<br />others 1990a) according to their storage behavior.<br />Recalcitrant seeds are those that undergo little, or no,<br />maturation drying and remain desiccation sensitive both during<br />development and after they are shed. The situation is, howev-<br />er, far more complex than this because of the wide range of<br />variability among recalcitrant seeds of different species and,<br />indeed, of individual species under different conditions (Ber-<br />jak and Pammenter 1997). Such seeds are shed hydrated, but<br />the water content can generally be anywhere in the range from<br />0.43 to 4.0 g g-1, which is 30 to 80 percent on a wet mass basis<br />(wmb). Shedding water content is partly species characteristic,<br />depending on the degree of dehydration that occurs late dur-<br />ing seed development; this has been suggested to be correlat-<br />ed with the degree of desiccation tolerance developed by indi-<br />vidual species (Finch-Savage 1996).<br />Recalcitrant seeds are not equally desiccation sensitive,<br />in that variable degrees of dehydration are tolerated depend-<br />ing on the species. This implies that the processes or mecha-<br />nisms (see below) that confer desiccation tolerance are vari-<br />ably developed or expressed in the nonorthodox condition. As<br />diverse mechanisms have been suggested to be involved in the<br />acquisition of desiccation tolerance and maintenance of the<br />integrity of dehydrated orthodox seeds, it should be appreci-<br />ated that any one of these may be absent, or present but inef-<br />fective, in recalcitrant seeds. Another important consideration<br />is that desiccation tolerance is probably controlled by the inter-<br />play of mechanisms or processes, and not by any one, acting in<br />isolation. Thus, the absence or incomplete expression of any fac-<br />tor proposed to confer dehydration tolerance could have pro-<br />found consequences on the ability of a seed species to withstand<br />a measure of dehydration below a particular level of hydration.<br />Differential desiccation sensitivity among recalcitrant<br />seeds of various species is clearly shown by their different<br />responses when subjected to the same drying regime–those of<br />some species tolerating only a slight degree of dehydration,<br />but others surviving to far lower water contents. There are also<br /> <br /><br /><br /><br /><br />marked differences in the rates at which water will be lost from<br />seeds of various species under the same dehydrating condi-<br />tions (Farrant and others 1989). Other factors, too, influence<br />the postharvest responses of recalcitrant seeds, e.g. develop-<br />mental status (Berjak and Pammenter 1997, Berjak and others<br />1992, Berjak and others 1993, Finch-Savage 1996, Finch-Sav-<br />age and Blake 1994) and chilling sensitivity (Berjak and Pam-<br />menter 1997).<br />In terms of desiccation sensitivity alone, therefore, it is<br />not merely that a seed species is recalcitrant, but rather, how<br />recalcitrant it is. This fact led to the proposal of a continuum of<br />recalcitrant seed behavior, from species that are highly desic-<br />cation–and probably also chilling–sensitive, to those that will<br />tolerate drying to the lowest water content still commensurate<br />with recalcitrant seed behavior and will also tolerate relatively<br />low temperatures (Farrant and others 1988).<br />The concept of a continuum of postharvest seed behav-<br />ior (that is, dependent on preshedding developmental events)<br />extends beyond the category of recalcitrant seeds. The contin-<br />uum grades from extreme desiccation-sensitive types through<br />the minimally recalcitrant types, to the intermediate seed<br />species that do not react adversely to low temperatures,<br />through those that are chilling sensitive when dehydrated<br />(Hong and Ellis 1996), and finally to orthodox seeds that will<br />tolerate less or more extreme dehydration (Vertucci and Roos<br />1990). It is also possible that there are seed species that behave<br />in a manner that characterizes them as lying between the hith-<br />erto-defined categories: recalcitrant, intermediate, and ortho-<br />dox. The idea of an extended continuum of seed behavior<br />from the most desiccation tolerant of orthodox species, to the<br />recalcitrant species that are most sensitive to even slight water<br />loss, embodies many properties of seeds and their responses<br />(Berjak and Pammenter 1994, 1997). It has its foundations in<br />an appreciation of the physiological status of seeds at various<br />water potentials (Vertucci 1993, Vertucci and Farrant 1995,<br />Vertucci and Roos 1990) and the properties of water at the var-<br />ious hydration levels corresponding to specified water poten-<br />tial ranges (Vertucci 1993, Vertucci and Farrant 1995). It is<br />more meaningful to consider seed responses to dehydration in<br />terms of water potential rather than water content, but, since<br />these two measures can be loosely correlated (Vertucci and<br />Farrant 1995), the more familiar water content terminology is<br />used here.<br />According to Vertucci and Farrant (1995): “Discrete<br />changes in metabolic activity with moisture content are hypoth-<br />esized to be associated with discrete changes in the physical<br />properties of water… Thus upon the loss of water with certain<br />properties, an essential function provided by [that] water is no<br />longer possible. A tissue that is not damaged by the removal of<br />a certain type of water has developed mechanisms to tolerate<br /> <br /><br /><br />138<br /> <br /><br /><br />Part I—Technical Chapters <br /><br /><br /><br /><br />or avoid that particular stress.” While the discussion that fol-<br />lows is not dependent on the reader’s appreciation of the dif-<br />ferences in the types of intracellular water, the basis of the<br />arguments presented is that sequential removal of water with<br />specific properties will have particular damaging effects on<br />seed tissues that are not possessed of the appropriate mecha-<br />nisms or processes to counteract that damage. We will, how-<br /> <br /><br /><br /><br /><br />additional properties that contribute to the ability of seeds to<br />withstand extreme dehydration are likely to be elucidated.<br /><br />INTRACELLULAR PHYSICAL<br />CHARACTERISTICS<br /><br />Vacuolation and Reserve Deposition<br /> <br />ever, focus on the mechanisms or processes themselves.<br />In 1957, Iljin had already identified one of the major require-<br />ments of cells of desiccation-tolerant plant material: the abili-<br />ty to withstand mechanical stress. Vacuole volume reduction,<br /> <br />MECHANISMS IMPLICATED IN<br />DESICCATION TOLERANCE<br /><br /><br /><br />It is most expedient to consider the processes or mechanisms<br />listed below, which might confer protection against desicca-<br />tion, and their deficiency or absence, which could contribute<br />to the relative degrees of desiccation sensitivity.<br /><br />• Intracellular physical characteristics such as<br />— reduction of the degree of vacuolation,<br />— amount and nature of insoluble reserves<br />accumulated,<br />— integrity of the cytoskeleton,<br />— conformation of the DNA, chromatin, and<br />nuclear architecture.<br />• Intracellular de-differentiation, which effectively results in<br />the minimization of surface areas of membranes and proba-<br />bly also of the cytoskeleton.<br />• “‘Switching off” of metabolism.<br />• Presence, and efficient operation of, antioxidant<br />systems.<br />• Accumulation and roles of putatively protective molecules,<br />including late embryogenic accumulating/abundant proteins<br />(LEA’s), sucrose and certain oligosaccharides, or galactosyl<br />cyclitols.<br />• Deployment of certain amphipathic molecules.<br />• An effective peripheral oleosin layer around lipid bodies.<br />• The presence and operation of repair mechanisms during<br />rehydration.<br /><br />In the discussion that follows comparisons are made, as<br />far as is possible, between desiccation-sensitive and orthodox<br />seeds, and of the status of the processes or mechanisms that<br />have been suggested to contribute to desiccation tolerance.<br />Although the interrelationships among them are far from being<br />resolved, these processes or mechanisms are those that have<br />been implicated to date in the acquisition and maintenance of<br />desiccation tolerance. However, it is important to realize that<br /> <br />whether by the shrinkage of the space occupied by these usu-<br />ally fluid-filled organelles or by their becoming filled with<br />insoluble reserve material, is one of the mechanisms that<br />would contribute to increased mechanical resilience of cells to<br />dehydration. This aspect was examined by Farrant and others<br />(1997) for (1) Avicennia marina, the highly recalcitrant seeds<br />of which can withstand very little dehydration either before or<br />after they are shed; (2) Aesculus hippocastanum, a temperate<br />recalcitrant species, the seeds of which overwinter in the hydrat-<br />ed condition during which the necessary stratification occurs<br />to facilitate germination the following spring; and (3) Phaseo-<br />lus vulgaris, a typical orthodox seed that attains a low water<br />content prior to shedding and is long-lived in this condition.<br />Avicennia marina seeds lose no water during develop-<br />ment, and are as sensitive to dehydration before shedding as<br />after abscission (Farrant and others 1992b). These seeds, at<br />best, are unable to surive water contents lower than 0.5 g g-1<br />(33 percent wmb). The vacuoles ultimately occupy almost 60<br />percent on average of the volume across the cells of all axis tis-<br />sues, and 90 percent of the cotyledonary cells when mature. At<br />no stage do either the axial or cotyledonary vacuoles contain<br />insoluble reserves, the little insoluble reserve material occur-<br />ring as plastid starch. Seeds of A. hippocastanum naturally<br />undergo a measure of dehydration during development,<br />accompanied by an increase in relative desiccation tolerance<br />(Tompsett and Pritchard 1993). The mature seeds are more<br />desiccation tolerant than those of A. marina, being able to<br />withstand dehydration to water contents in the range of 0.42<br />to 0.25 g g -1 (30 to 20 percent wmb). Vacuoles ultimately con-<br />stitute only a small fraction of the intracellular volume, partic-<br />ularly in the axis cells at maturity. The cotyledonary cells con-<br />tain many large, starch-filled plastids and protein bodies and<br />are considerably less vacuolate than those of A. marina. In P.<br />vulgaris seeds, which are orthodox and able to tolerate low<br />water contents, vacuolar volume is reduced to an insignificant<br />proportion in axis cells, and vacuoles in cotyledonary cells<br />accumulate an amorphous, presumably insoluble, material.<br />The differential degree of vacuolation and insoluble reserve<br />deposition among the three species, in both developing and<br /><br /><br />Chapter 4: Orthodox and Recalcitrant Seeds <br /><br /><br /><br /><br />mature seeds, correlates with their degree of desiccation sensi-<br />tivity. This is in accord with the concept that a high degree of<br />vacuolation can lead to lethal mechanical damage upon dehy-<br />dration (Farrant and others 1997).<br /><br />Reaction of the Cytoskeleton<br /><br />The cytoskeleton, the major components of which are micro-<br />tubules and microfilaments, is not only an integrated intracel-<br />lular support system, it also plays a major role in imposing<br />organization on the cytoplasm and also the nucleus. Micro-<br />tubules consist of polymerized a -tubulin, and microfilaments<br />are composed of F-actin, which is a polymer of G-actin. We<br />are presently investigating the status of the actin microfila-<br />ments in hydrated and variously dehydrated embryonic axes of<br />seeds of Quercus robur, a temperate recalcitrant species. In the<br />hydrated state, there is an extensive microfilamentous network<br />in the cells of the root tip, which becomes dismantled as the<br />seeds are increasingly dehydrated–a feature that is expected<br />for orthodox seeds as well. In such desiccation-tolerant seeds,<br />orderly reassembly of the elements of the cytoskeleton accom-<br />panies imbibition, but once the water content falls to dam-<br />agingly low levels in Q. robur, the microfilaments are not<br />reassembled when the seeds are subsequently rehydrated<br />(Mycock and others 2000). The resultant lack of the intracel-<br />lular support and structural organization afforded by the<br />cytoskeleton would obviously be a major damaging factor upon<br />rehydration of recalcitrant seeds. Additionally, certain cytoma-<br />trical (cytoplasmic) enzyme systems exist as multienzyme par-<br />ticles in plant cells (Hrazdina and Jensen 1992), the formation<br />of which could occur because of the binding of key or anchor<br />enzymes to the microfilaments of the cytoskeleton, as illustrat-<br />ed for glycolysis by Masters (1984). Thus, failure of the<br />cytoskeleton to reassemble following deleteriously low levels<br />of dehydration would have physiological as well as structural<br />consequences in the cells of desiccation-sensitive seed tissues.<br /><br />DNA, Chromatin, and<br />Nuclear Architecture Conformation<br /><br />Maintenance of the integrity of the genetic DNA material in<br />the desiccated condition in orthodox seeds, and/or its rapid<br />repair when seeds are rehydrated, is considered to be a funda-<br />mental requirement for desiccation tolerance. There is, how-<br />ever, little information on which to draw. DNA assumes dif-<br />ferent conformational states depending on water activity and,<br />although this has not yet been demonstrated for seeds, it is<br />considered that as water is lost (i.e. water activity is lowered)<br />such conformational changes will occur (Osborne and Boubri-<br />ak 1994). According to information reviewed by those authors,<br /> <br /><br /><br /><br /><br />there is an increase in the number of base pairs per turn of the<br />DNA helix as water is lost from the individually hydrated<br />phosphate groups, and water bridges are formed instead as the<br />conformation changes from the B to the Z form. Osborne and<br />Boubriak (1994) have suggested that protein glycation (i.e. the<br />nonenzymic addition of reducing sugars to [i.a. ] histone pro-<br />teins) is likely to occur, which could increase the incidence of<br />DNA conformations appropriate to the dehydrated state.<br />Those authors also discuss the possibility of nonenzymic<br />methylation of cytosine occurring, which would favor the Z-<br />form of the DNA.<br />However, besides the postulated necessity of conforma-<br />tional changes in the DNA occurring as desiccation-tolerant<br />material is dehydrated, the structure of the chromatin itself<br />must also be stabilized. The highly condensed state of the<br />chromatin in dry, orthodox seeds (e.g. Crévecoeur and others<br />1976, Sargent and others 1981), which is reversed at the stage<br />in germination when desiccation sensitivity ensues (Deltour<br />1985), is thought to be a visible manifestation of its stabilized<br />condition. A major factor in chromatin stabilization in the dry<br />state in orthodox seeds might be the change in the H1-his-<br />tone:nucleosome ratio to 2:1 from the 1:1 ratio that typifies the<br />hydrated condition (Ivanov and Zlatanova 1989).<br />Nuclear architecture is a further factor that is probably<br />involved in chromatin stability. The structural framework of<br />the nucleus has been convincingly demonstrated for plant cells<br />and is based on intermediate-type filaments called lamins<br />(Moreno Díaz de la Espina 1995). The nucleoskeleton, organ-<br />ized into the lamina (underlying and connected to the inner<br />surface of the nuclear envelope) and matrix (ramifying through-<br />out the nucleus) is suggested to support and localize the chro-<br />matin in discrete domains, imposing the topological organiza-<br />tion and coordination of intranuclear processes (Moreno Díaz<br />de la Espina 1995). It is implicit that during dehydration and<br />in the desiccated state of orthodox seeds, orderly reorganiza-<br />tion of the nucleoskeleton should occur with its restitution as<br />a functional framework upon rehydration.<br />While little is known about the effects of dehydration on<br />the DNA, chromatin, and nuclear architecture in desiccation-<br />sensitive seeds, their stability in the dehydrated state clearly<br />must be a prerequisite for desiccation tolerance. Maintenance<br />of the integrity of the nucleus as a whole, and the genome in<br />particular, may be imperfectly expressed, or the ability for this<br />may even be totally lacking, in recalcitrant seeds. (For a fuller<br />account of some of these aspects, see Leprince and others<br />(1995) and Pammenter and Berjak (1999)). What is equally<br />likely is that DNA repair mechanisms themselves are inade-<br />quate to restitute damage caused by dehydration of desicca-<br />tion-sensitive seeds (see below).<br /> <br /><br /><br />140<br /> <br /><br /><br />Part I—Technical Chapters <br /><br /><br /><br /><br />INTRACELLULAR<br />DE-DIFFERENTIATION<br /><br />De-differentiation, a characteristic of maturing desiccation-<br />tolerant seeds, is essentially a means by which intracellular<br />structures are simplified and minimized (reviewed by Vertuc-<br />ci and Farrant 1995), which strongly suggests that membranes<br />and cytoskeletal elements are vulnerable to dehydration. This<br />phenomenon is reversed in orthodox seeds when water is<br />taken up early during germination (Bewley 1979, Dasgupta<br />and others 1982, Galau and others 1991, Klein and Pollock<br />1968, Long and others 1981).<br />An examination of the quantitative and qualitative status<br />of mitochondria in seeds of Avicennia marina, Aesculus hip-<br />pocastanum, and Phaseolus vulgaris showed that the propor-<br />tion of cell volume occupied by these organelles was highest in<br />A. marina, which is very desiccation sensitive, and substantial-<br />ly less for A. hippocastanum, which is in keeping with its less<br />recalcitrant nature. In P. vulgaris, mitochondria occupied a sig-<br />nificantly smaller proportion of the cell volume, even preced-<br />ing the onset of maturation drying (Farrant and others 1997).<br />Also, the mitochondria occupied a far greater proportion of<br />the cell volume in the axis meristems of the two recalcitrant<br />species than in the orthodox species, P. vulgaris. There were<br />also marked differences in the structural complexity of the<br />mitochondria among these three species: A. marina and A. hip-<br />pocastanum, had well-developed cristae and a structure that<br />was generally typical of an active, hydrated plant tissue; while<br />in P. vulgaris, the mitochondria were almost completely de-dif-<br />ferentiated even at tissue water contents comparable to those<br />of the recalcitrant species at shedding (Farrant and others<br />1997). It thus seems that retention of organelles in the highly<br />differentiated state is a major factor in the desiccation sensi-<br />tivity of recalcitrant species, whereas the ability for ordered<br />de-differentiation is, in fact, a prerequisite for seed survival in<br />the dehydrated state.<br />There has long been uncertainty as to whether dehydra-<br />tion causes de-differentiation, or this intracellular minimiza-<br />tion actually precedes the initiation of maturation drying (e.g.<br />Vertucci and Farrant 1995). However, the observations on P.<br />vulgaris reported by Farrant and others (1997), indicating that<br />mitochondrial de-differentiation occurs, and that respiratory<br />rate declines markedly (see also below) before maturation dry-<br />ing, support the idea that substantial qualitative and quantita-<br />tive change actually occurs in advance of water loss.<br /><br />“SWITCHING OFF” OF METABOLISM<br /><br />Electron transport, albeit at a low level, has been recorded for<br />dehydrated plant tissues, and respiration is measurable even at<br /> <br /><br /><br /><br />seed water contents as low as 0.25 g g-1 [20 percent, wmb]<br />(Vertucci 1989, Vertucci and Farrant 1995). However, in the<br />water content range 0.45 to 0.25 g g (30 to 20 percent<br />[wmb]), unbalanced metabolism may lead to the generation,<br />and essentially uncontrolled activity, of free radicals (Finch-<br />Savage and others 1994a, Hendry 1993, Hendry and others<br />1992, Leprince and others 1990b, Vertucci and Farrant 1995).<br />It is therefore imperative that, during maturation drying, des-<br />iccation-tolerant seeds be able to pass through this water con-<br />tent range with the minimum of damage. The efficient opera-<br />tion of antioxidant systems (Leprince and others 1993, Pun-<br />tarulo and others 1991), as well as the “switching off” of<br />metabolism, would reduce such damage. Rogerson and<br />Matthews (1977) recorded that a sharp decline in respiratory<br />substrates precedes, and presumably causes, the fall in respi-<br />ratory rate which, they suggested, is an essential event enabling<br />an orthodox seed to withstand rapid loss of water. The obser-<br />vations of Farrant and others (1997), indicating that a decline<br />in respiratory rate occurs while mitochondria become sub-<br />stantially de-differentiated prior to maturation drying in the<br />orthodox seeds of Phaseolus vulgaris, support the data and<br />suggestions of Rogerson and Matthews (1977).<br />In desiccation-sensitive seeds, lethal damage occurs in<br />the water content range 0.45 to 0.25 g g1 (Vertucci and Farrant<br />1995) and, in some species, at considerably higher levels<br />(Pammenter and others 1993). Death of relatively hydrated<br />recalcitrant seeds (at c. 0.7 g g-1 , or higher [ 40 percent, wmb])<br />occurs when water is lost slowly. However, rapid dehydration<br />rates allow survival to lower water contents (Farrant and oth-<br />ers 1985). This observation led initially to the use of relatively<br />rapid air-drying of excised embryonic axes to facilitate cryos-<br />torage (Normah and others 1986, Pritchard and Prendergast<br />1986) and later to the development of the flash-drying tech-<br />nique (Berjak and others 1990), by which the axes are dehy-<br />drated much more rapidly.<br />Flash-dried axes are not desiccation tolerant; on the con-<br />trary, they will not survive for longer than a day or two at best,<br />under ambient conditions (Walters and others 2001) although<br />they may be cryostored successfully (Wesley-Smith and others<br />1992). The desiccation sensitivity of recalcitrant material is the<br />outcome of the fact that the axes (seeds) are actively metabol-<br />ic, and the success of very rapid dehydration is that it mini-<br />mizes the effects of this metabolism. This important point<br />about drying rate is discussed in detail later.<br />Damage occurring in conjunction with unbalanced<br />metabolism at these relatively high water contents should not<br />be confused with desiccation damage in the strict sense. The<br />latter describes the damage that occurs when water that is<br />required to maintain the integrity of intracellular structures is<br />removed (Walters and others 2001). Desiccation damage sensu<br /><br /><br />Chapter 4: Orthodox and Recalcitrant Seeds <br /><br /><br /><br /><br />stricto is the consequence of removing (any, or some, depend-<br />ing on the species) structure-bound, nonfreezable water (Pam-<br />menter and others 1991, Walters and others 2001). Lethal<br />damage occurs upon loss of this water, even if flash-drying has<br />successfully maintained axis viability to, or close to, this level<br />of hydration (Pammenter and others 1991).<br />Another critical aspect of ongoing metabolism is cell<br />cycling. The cell cycle describes the nuclear DNA content as<br />2C in cells that are not preparing for nuclear division, and as<br />4C in cells in which DNA replication has occurred, where the<br />constant, C, denotes the DNA content of the haploid condi-<br />tion. During the cell cycle four distinct phases can be identi-<br />fied, viz. the G1 phase (2C), which is followed by the S phase,<br />during which DNA replication occurs; after this the cells enter<br />the G2 phase, during which the amount of DNA remains dou-<br />bled (i.e. 4C) as a result of events in the S phase, and this is fol-<br />lowed by the phase known as G2M, when mitosis reduces the<br />DNA content to the 2C level typical of somatic cells in the<br />next G1 phase. Brunori (1967) found that in orthodox Vicia<br />faba seeds, most of the cells were arrested in G1, and that<br />DNA replication was one of the first events to be curtailed as<br />the embryo cells lost water. S-phase replication is resumed<br />only after several hours of imbibition, when water again<br />becomes available to postharvest, orthodox seeds, as shown by<br />Sen and Osborne (1974) for Secale cereale (rye): as soon as<br />replication to 4C values occurs and the cells enter G2M, des-<br />iccation tolerance is lost.<br />In the the highly recalcitrant seeds of Avicennia marina,<br />there is only the most transient arrest of DNA replication in<br />root primordia (meristems) of Avicennia marina lasting no<br />more than 24 hours around shedding. This is the time when<br />the seeds (although highly desiccation sensitive) are relatively<br />most tolerant of water loss and least active. Ongoing cell<br />cycling is associated with marked desiccation sensitivity of the<br />DNA. When only 16 to 18 percent of the total water is lost<br />from the A. marina material, there is a reduction of 70 to 80<br />percent in the nuclei that will incorporate thymidine, and after<br />a 22-percent water loss, damage of the DNA cannot be<br />repaired even when water is made freely available. Ongoing<br />cell cycling, therefore, is another manifestation of the fact that<br />metabolism is not “switched off,” at least in these highly recal-<br />citrant seeds, which is considered to be a major factor account-<br />ing for their desiccation sensitivity. In related work on the tem-<br />perate recalcitrant species Acer pseudoplatanus, however, cell<br />cycling was found to be arrested, with over 60 percent of the<br />cells in the 2C state (Finch-Savage and others 1998). Howev-<br />er, seeds of A. marina are poised for immediate germination,<br />while those of A. pseudoplatanus are dormant, requiring cold<br />stratification before they will germinate. For seeds of<br />Azadirachta indica, recorded as showing intermediate behav-<br /> <br /><br /><br /><br /><br />ior, the 2C DNA level has been reported as occurring to the<br />virtual exclusion of 4C (Sacandé and others 1997). These dis-<br />parate results on the status of the cell cycle in three nonortho-<br />dox seed species serve to highlight the fact that different fac-<br />tors may contribute to the nature, and differing degrees, of<br />desiccation sensitivity.<br /><br />PRESENCE AND<br />EFFICIENT OPERATION OF<br />ANTIOXIDANT SYSTEMS<br /><br />A range of antioxidant processes operate in orthodox seeds<br />(e.g. Hendry 1993, Leprince and others 1993), and the role of<br />such processes under conditions of water deficit and desicca-<br />tion stress in plants has been reviewed by McKersie (1991) and<br />Smirnoff (1993). As discussed above, it is particularly in the<br />water content range from 0.45 to 0.25 g g -1 (30 to 20 percent,<br />wmb), that unregulated metabolic events resulting in the first<br />wave of free-radical generation are likely to occur (Vertucci<br />and Farrant 1995). This implies that antioxidant systems (i.e.<br />free-radical scavenging systems) should be maximally effective<br />during maturation drying of orthodox seeds, and again when<br />seeds take up water upon imbibition.<br />Reviews of metabolic damage associated with dehydra-<br />tion of recalcitrant seeds highlight the idea that free-radical<br />generation may well be a major injurious factor (Berjak and<br />Pammenter 1997; Côme and Corbineau 1996a, 1996b; Smith<br />and Berjak 1995), particularly because protective mechanisms<br />appear to become impaired under conditions of water stress<br />(Senaratna and McKersie 1986, Smith and Berjak 1995). Rapid<br />formation of free radicals and decreasing activity of antioxi-<br />dant systems have been reported as occurring during dehydra-<br />tion of the seeds of the temperate recalcitrant species Quercus<br />robur (Finch-Savage and others 1993). Lipid peroxidation,<br />which is a major consequence of uncontrolled free-radical gen-<br />eration, with the ultimate accumulation of a stable free radical<br />in the embryonic axes, has been shown to accompany dehy-<br />dration of the seeds of three temperate, recalcitrant species—<br />-Q. robur, Castanea sativa, and Aesculus hippocastanum (Finch-<br />Savage and others 1994a)–and free radical formation has been<br />reported to accompany viability loss in seeds of the highly<br />recalcitrant, tropical species Shorea robusta (Chaitanya and<br />Naithani 1994). While hydroperoxide formation has been<br />shown to accompany dehydration at a range of temperatures<br />of the recalcitrant seeds of Zizania palustris, significantly more<br />was produced at 37 °C than at 25 oC, and tetrazolium tests<br />revealed that viability was severely affected by water loss at the<br />higher temperature (Ntuli and others 1997).<br />From the evidence reviewed above, there is no doubt<br />that damage ascribable to uncontrolled free-radical generation<br /> <br /><br /><br />142<br /> <br /><br /><br />Part I—Technical Chapters <br /><br /><br /><br /><br />occurs during dehydration in the recalcitrant seeds of a range<br />of species that show differing degrees and manifestations of<br />nonorthodox behavior. This implies not only that free radicals<br />are produced as a consequence of water stress in these desic-<br />cation-sensitive seeds, but also that antioxidant systems are<br />ineffective at curbing them. Together, then, these factors must<br />be seriously considered as constituting one of the major caus-<br />es of desiccation sensitivity.<br /><br />ACCUMULATION AND ROLES OF<br />PUTATIVELY PROTECTIVE<br />MOLECULES<br /><br />Late Embryogenic Accumulating/Abundant<br />Proteins (LEA’s)<br /><br />LEA’s (Galau and others 1986) comprise a set of hydrophilic,<br />heat-resistant proteins associated with the acquisition of des-<br />iccation tolerance in developing orthodox seeds (Galau and<br />others 1991 reviewed by Bewley and Oliver 1992, Kermode<br />1990, Ried and Walker-Simmons 1993). Their synthesis appears<br />to be associated with the high ABA levels that peak during the<br />later stages of seed development (Kermode 1990). The charac-<br />teristics of LEA’s and the conditions under which they appear<br />have led to suggestions that they function as protectants, per-<br />haps stabilizing subcellular structures in the desiccated condi-<br />tion (Close and others 1989, Dure 1993, Lane 1991).<br />The position of LEA’s (or dehydrin-like proteins, as they<br />may be termed) in nonorthodox seeds appears at first sight to<br />be anomalous, as some species do not express these proteins<br />while others express them to variable extents. Seeds of Avi-<br />cennia marina, which are extremely desiccation sensitive,<br />appear not to express LEA’s at all (Farrant and others 1992a).<br />In contrast, seeds of Zizania palustris (North American wild<br />rice), which are recalcitrant (Vertucci and others 1994) but<br />show differential responses to dehydration depending on tem-<br />perature (Kovach and Bradford 1992a, Ntuli and others 1997),<br />do express this type of protein (Bradford and Chandler 1992,<br />Still and others 1994). Dehydrin-like proteins were shown to<br />be expressed in a range of temperate, recalcitrant species<br />(Finch-Savage and others 1994b, Gee and others 1994), but<br />the absence of such proteins correlated with low ABA levels<br />was found to characterize the mature, recalcitrant seeds of 10<br />tropical, wetland species (Farrant and others 1996). Those<br />authors showed the presence of dehydrin-like proteins in other<br />temperate and tropical recalcitrant (nonwetland) species, and<br />suggested that their occurrence may be habitat-related, per-<br />haps also providing protection against low-temperature stress.<br />In a comparative study on mature seeds of two tropical tree<br />species, neither of which occurs in wetlands, dehydrin-type<br /> <br /><br /><br /><br /><br />proteins were absent in Trichilia dregeana, while accumulating<br />in Castanospermum australe (Han and others 1997). The<br />immature seeds and the seedlings of these two species were<br />shown to differ in terms of production of such proteins in<br />response to stresses imposed by dehydration, application of<br />ABA, or exposure to cold, with T. dregeana not responding by<br />the production of these putatively protective proteins (Han<br />and others 1997).<br />Thus, it seems that the ability to express LEA’s or dehy-<br />drin-type proteins cannot be taken as an indication that the<br />seeds of a particular species will or will not withstand dehy-<br />dration. This indicates clearly that desiccation tolerance must<br />be the outcome of the interplay of more than one (and proba-<br />bly many) mechanisms or processes. Details of this, particu-<br />larly pertaining to LEA’s/dehydrins, sugars, and various stress-<br />es, have been reviewed by Kermode (1997). However, the vari-<br />able expression of LEA’s/dehydrins in recalcitrant seeds on a<br />species basis may, in association with the presence or absence<br />of other factors, account for the degree of nonorthodox behav-<br />ior exhibited under a particular set of circumstances.<br /><br />Sucrose, Oligosaccharides, or<br />Galactosyl Cyclitols<br /><br />The possible role(s) of nonreducing sugars in relation to des-<br />iccation tolerance in seeds has been extensively reviewed (e.g.<br />by Berjak and Pammenter 1997, Horbowicz and Obendorf<br />1994, Obendorf 1997, Vertucci and Farrant 1995). Accumula-<br />tion of nonreducing sugars, particularly of the raffinose series<br />(Blackman and others 1992, Koster and Leopold 1988, Lep-<br />rince and others 1990a) and/or galactosyl cyclitols (Horbowicz<br />and Obendorf 1994, Obendorf 1997) has been implicated in<br />the acquisition and maintenance of the desiccated state in<br />orthodox seeds, generally in two major ways. These are in<br />terms of the “Water Replacement Hypothesis” (Clegg 1986,<br />Crowe and others 1992) and vitrification, otherwise referred to<br />as glassy state formation (Koster and Leopold 1988, Leopold<br />and others 1994, Williams and Leopold 1989).<br />Orthodox seed maturation invariably seems to be<br />accompanied by the accumulation of nonreducing oligosac-<br />charides which coincides with the reduction of monosaccha-<br />rides, and maintenance of the desiccated state is associated<br />with high levels of sucrose and other oligosaccharides. Evi-<br />dence for the replacement of membrane-associated water (the<br />Water Replacement Hypothesis, i.e. the replacement of water<br />by sucrose to maintain lipid head-group spacing, thereby pre-<br />venting gel-state transformation) is equivocal, and a recent cri-<br />tique questions its relevance in the desiccated state of ortho-<br />dox seeds (Hoekstra and others 1997). However, the role of<br />sucrose in the formation of intracellular glasses (i.e. vitrifica-<br /><br /><br />Chapter 4: Orthodox and Recalcitrant Seeds <br /><br /><br /><br /><br />tion) is more convincing. The metastable, glassy state occurs at<br />low water contents in seeds, when sucrose and certain oligosac-<br />charides or galactosyl cyclitols form high-viscosity, amor-<br />phous, super-saturated solutions (Obendorf 1997). The occur-<br />rence of glasses is held to impose a stasis on intracellular reac-<br />tivity, protecting macromolecules against denaturation and<br />possibly preventing or minimizing liquid crystalline gel phase<br />transformations of the lipid bilayer of membranes (e.g.<br />Leopold and others 1994).<br />Walters and others (1997) have suggested that a signifi-<br />cant proportion of the sugars may be tightly associated with<br />LEA’s–these complexes acting to control and optimize the rate<br />of water loss during dehydration of orthodox seeds. It should<br />be noted, however, that this should not obviate the participa-<br />tion of either the LEA’s or the sugars in the maintenance of<br />orthodox seed viability in the desiccated state.<br />The formation of intracellular oligosaccharides occurs at<br />the expense of monosaccharides, and confers the advantage<br />that immediately available respiratory substrates are removed<br />(Koster and Leopold 1988, Leprince and others 1992, Roger-<br />son and Matthews 1977). This would serve to reduce the spec-<br />trum of damaging reactions that can occur as orthodox seeds<br />pass through critical water content ranges favoring unbalanced<br />metabolism, during maturation drying (see “Switching off” of<br />metabolism, above).<br />Whatever the role(s) of sucrose and oligosaccharides or<br />galactosyl cyclitols may be in orthodox seeds, seeking parallels<br />for desiccation-sensitive seeds is entirely inappropriate. While<br />sucrose and other oligosaccharides are produced in some of<br />the few recalcitrant seed species that have been assayed (Far-<br />rant and others 1993, Finch-Savage and Blake 1994), glass for-<br />mation will occur only at water contents well below the lethal<br />limit. When recalcitrant seeds are dehydrated under ambient<br />conditions (which is what would occur in the natural habitat),<br />they lose viability at relatively high water contents–in the<br />region of 0.7 g (or more) water per g dry mass [ 40 percent,<br />wmb] (Pammenter and others 1991), which are far higher that<br />those required for glass formation to occur (Bruni and<br />Leopold 1992, Leopold and others 1994, Sun and others 1994,<br />Williams and Leopold 1989). The same argument holds if<br />water replacement by sugars is an operative phenomenon in<br />orthodox seeds; this too would occur only at water contents of<br />0.3 g per g dry material (Hoekstra and Van Roekel 1988),<br />which is well below the lethal limit for slowly drying recalci-<br />trant seeds.<br />The one involvement of sugars in the variable desicca-<br />tion sensitivity of recalcitrant seeds might be via the mecha-<br />nism suggested by Walters and others (1997) for maturing<br />orthodox seeds, viz. the modulating effect of sugar/LEA com-<br />plexes on dehydration rate. Very marked variability occurs in<br /> <br /><br /><br /><br /><br />the rate at which recalcitrant seeds of different species lose<br />water under the same conditions (Berjak and Pammenter<br />1997, Farrant and others 1989) and it is possible that the sig-<br />nificance of sugars and LEA’s in embryos of recalcitrant seeds<br />of some species lies in the modulation of the drying rate by<br />complex formation. Walters and others (1997) have also sug-<br />gested that LEA proteins in temperate recalcitrant seeds may<br />play a role in their survival during overwintering.<br /><br />DEPLOYMENT OF CERTAIN<br />AMPHIPATHIC MOLECULES<br /><br />It has been suggested that partitioning of endogenous amphi-<br />pathic molecules (amphipaths) into membranes upon water<br />loss may be a prerequisite for desiccation tolerance (Golovina<br />and others 1998). Those authors have presented evidence of<br />the movement during dehydration of both introduced, apolar<br />spin probes and endogenous amphipaths into the bilayer of<br />desiccation-tolerant pollen. This process, which was complete<br />after dehydration to the relatively high water content of 0.6 g<br />per g dry mass (37 percent, wmb), was reversed during rehy-<br />dration, when the amphipaths repartitioned to the cytomatrix<br />(aqueous cytoplasm). This reverse movement was suggested to<br />account for the transient leakage that is invariably observed<br />when desiccation-tolerant material (pollen and seeds) is<br />imbibed from the dry state (Golovina and others 1998).<br />The partitioning of amphipathic molecules into the<br />bilayer was suggested by those authors as serving to maintain<br />the integrity of membranes in the dry state in desiccation-tol-<br />erant organisms, by substantially lowering the water content at<br />which the phase change of membrane lipids occurs. Liquid<br />crystalline to gel phase changes in membranes are well docu-<br />mented in response to dehydration, but the essential property<br />for desiccation tolerance is that they must be reversible,<br />reestablishing the membranes in a functional condition upon<br />rehydration (Hoekstra and others 1992). This demands that<br />integral membrane proteins retain their position in the desic-<br />cated state, a role that might also be ascribed to the amphi-<br />pathic molecules.<br />If the partitioning of amphipaths into membranes is<br />established as a universal phenomenon occurring during dehy-<br />dration of orthodox seeds, it is possible that they are absent or,<br />if present, incompletely functional or nonfunctional in desic-<br />cation-sensitive seeds. Dehydration of the embryos from<br />recalcitrant Camellia sinensis seeds was found to induce a<br />phase change in membrane lipids, which was reversible, but<br />the proteins were irreversibly affected (Sowa and others 1991).<br />It may be significant that at a water content of 0.6 g g-1 , when<br />amphipath partitioning has been observed to be complete<br />(Golovina and others 1998), slowly dried recalcitrant seeds,<br /> <br /><br /><br />144<br /> <br /><br /><br />Part I—Technical Chapters <br /><br /><br /><br /><br />and even the flash-dried axes of certain species, will have lost<br />viability (Pammenter and others 1991, 1993; also see below).<br />In highly desiccation-sensitive recalcitrant seeds, it is possible<br />that phase changes of the membrane bilayers might not be<br />reversible, for example, if nonbilayer structures or hexagonal<br />phases result (reviewed by Vertucci and Farrant 1995). Parti-<br />tioning of endogenous amphipaths into the bilayer upon dehy-<br />dration is unlikely to act in isolation; thus, even if such mole-<br />cules are present in cells of recalcitrant seeds, they may well<br />depend on another mechanism or process to achieve their<br />reversible migration.<br /><br />THE POSSIBLE ROLE<br />OF OLEOSINS<br /><br />The term oleosin refers to a unique protein type that surrounds<br />the lipid (oil) droplets in plant cells (Huang 1992). Oleosins<br />have a central, hydrophobic domain that interacts with the<br />periphery of the lipid, and an amphipathic N-terminal domain<br />that, with the C-terminal domain, facilitates interaction with<br />the aqueous cytomatrix. The oleosin boundary of lipid bodies<br />allows these hydrophobic masses to be accommodated as dis-<br />crete entities in the aqueous cytomatrix under hydrated con-<br />ditions, and it has been suggested that their role during dehy-<br />dration prevents the bodies from coalescing in desiccation-tol-<br />erant seeds (Leprince and others 1997).<br />Leprince and others (1997) recorded a lack (or inade-<br />quate amount) of oleosins in desiccation-sensitive seeds of<br />some species, and although little obvious change in the integri-<br />ty of the bodies as a consequence of dehydration was<br />observed, rehydration appeared to have deleterious effects on<br />their stability. Coalescence of lipid bodies is a common abnor-<br />mality accompanying deterioration, even in cells of stored,<br />orthodox seeds (Smith and Berjak 1995). Although the effects<br />of fungi associated with both stored orthodox and recalcitrant<br />seeds in bringing about lipid body coalescence cannot be ruled<br />out, the occurrence of this phenomenon could well be, at least<br />partly, a consequence of some deficiency in desiccation-sensi-<br />tive seeds. In view of the findings of Leprince and others<br />(1997), the deficiency of an adequate oleosin sheath around<br />the lipid bodies may underlie the inherent instability of these<br />organelles during rehydration following damaging levels of<br />desiccation of some recalcitrant seeds. However, it must be<br />stressed that the presence of fully functional oleosins cannot,<br />in itself, account for desiccation tolerance. Rather, it must be<br />viewed as one of the mechanisms contributing to the spectrum<br />of properties necessary if orthodox seeds are to survive<br />extreme dehydration.<br /> <br /><br /><br /><br /><br />THE PRESENCE AND OPERATION<br />OF REPAIR MECHANISMS<br />DURING REHYDRATION<br /><br />There is both indirect and direct evidence that repair mecha-<br />nisms do come into play when dry orthodox seeds are rehy-<br />drated. For example, seeds that have been stored under<br />adverse conditions, but are still 100-percent viable, typically<br />show a lag before there are visible signs of germination, during<br />which it is commonly accepted that repair processes are taking<br />place. Ultrastructural studies on maize seeds have provided<br />evidence supporting this contention, where mitochondrial<br />repair was observed during the lag period (Berjak and Villiers<br />1972). Studies on rye seeds have shown that even in the dry<br />state there is progressive deterioration of the DNA as a result<br />of endo- and exonuclease activity during storage (Elder and<br />others 1987), which cannot be repaired until the seeds are<br />rehydrated (Boubriak and others 1997).<br />Much of the evidence for the operation of repair process-<br />es during rehydration comes from osmopriming experiments<br />on low-vigor seeds. This process involves controlled rehydra-<br />tion to the end of phase II, which achieves a hydration level<br />that facilitates repair but precludes germination proper (Bray<br />1995, Bray and others 1993). Those authors have shown that<br />replacement of damaged rRNA occurs, and lesions in the DNA<br />and protein-synthesizing systems are repaired, during priming.<br />It is generally agreed that free radical generation (see<br />above) continues in air-dried orthodox seeds during storage<br />(reviewed by Smith and Berjak 1995) and the ensuing damage<br />obviously must be repaired on rehydration, arguing strongly<br />for the presence and efficient operation of antioxidant systems<br />at this stage. During dehydration of desiccation-sensitive seeds<br />and seedlings, however, such systems have been shown to fail<br />(Hendry and others 1992, Leprince and others 1992) and are<br />assumed to remain ineffective when water is once again pro-<br />vided (Côme and Corbineau 1996a, 1996b).<br />When recalcitrant seeds or axes excised from such seeds<br />are subjected to nonlethal dehydration, it is generally observed<br />that there is an increase in the time taken for the onward<br />growth of germination, which might be interpreted as facili-<br />tating repair. However, this is likely to be strictly limited; pres-<br />ent studies have shown that after 22 percent of the water is lost<br />from hypocotyl tips of Avicennia marina, dehydration-associ-<br />ated DNA damage can no longer be repaired when water is<br />once again provided. DNA instability to dehydration is also<br />shown by seedlings produced from orthodox seeds, once they<br />have reached the stage when desiccation tolerance has been<br />lost (Boubriak and others 1997).<br />Very little work that targets the aspect of possible repair<br />of mature, dehydration-damaged recalcitrant seeds has yet<br /><br /><br />Chapter 4: Orthodox and Recalcitrant Seeds <br /><br /><br /><br /><br />been done. It is presently tacitly assumed that the necessary<br />repair systems are present, but are themselves damaged by<br />dehydration beyond certain limits–limits that might vary among<br />seed species of markedly differing desiccation sensitivity. How-<br />ever, this aspect requires considerable investigation to obtain<br />both qualitative and quantitative data to clarify the situation.<br /><br /><br /><br /><br />DRYING RATE - A VITAL FACTOR<br />IN DETERMINING THE<br />DEGREE OF DEHYDRATION<br />THAT WILL BE TOLERATED<br /><br /><br /><br />We now know that much confusion has occurred in compara-<br />tive work on individual species of recalcitrant seeds because of<br />conflicting data regarding “critical water contents,” below<br />which viability will be lost. This is because the dimensions of<br />the time taken for water to be lost, or the temperature at which<br />the drying experiments were carried out, have been ignored.<br />While the effects of temperature will not presently be dis-<br />cussed, there are several publications focused on the seeds of<br />Zizania spp. which show that this parameter can have very<br />marked effects on the outcome of drying regimes and/or opti-<br />mal storage water contents (Kovach and Bradford 1992b;<br />Ntuli and others 1997; Vertucci and others 1994, 1995). The<br />effect of the maturity status of the seeds–which is often<br />extremely difficult to ascertain for recalcitrant types–also has<br />significant effects on the degree of dehydration that will be tol-<br />erated (reviewed by Berjak and Pammenter 1997, Finch-Sav-<br />age 1996) but also will not be taken further.<br />The aspect of the time taken for water to be lost is a vari-<br />able that has been identified as having profound effects on the<br />degree of dehydration that desiccation-sensitive seed material<br />will tolerate. The more rapidly dehydration can be achieved,<br />the lower is the water content to which the seeds or axes can<br />be dried without damage accumulation that culminates in via-<br />bility loss. This is particularly marked when excised axes are<br />dried (Berjak and others 1993; Normah and others 1986; Pam-<br />menter and others 1991, 1993). Very rapid drying of excised<br />recalcitrant axes (flash-drying) facilitates nonlethal dehydra-<br />tion to water contents in the region of 0.4 to 0.25 g g-1 dm,<br />which is close to the hydration level where all the water is non-<br />freezable (generally structure-associated), although tolerance<br />to such low water contents is not invariably the case (Pam-<br />menter and others 1993). It must be noted, however, that such<br />rapid drying does not mean that the seed tissues are potential-<br />ly desiccation tolerant; rather, the faster dehydration can be<br />achieved, the less the time during which the axes are in the<br /> <br /><br /><br /><br /><br />water content range that permits damaging, potentially lethal,<br />aqueous-based reactions to occur. As discussed below, these<br />are the processes that, given sufficient time, will cause viabili-<br />ty loss at relatively high water contents when the tissues are<br />dehydrated slowly (Berjak and others 1989, 1993; Pammenter<br />and others 1998; Pritchard 1991). Far from actually being des-<br />iccation tolerant, axes from recalcitrant seeds will survive only<br />for very short periods (hours to a day or two), at the lowest<br />water contents attainable (Walters and others 2001).<br />Marked effects of drying rate on whole seeds are gener-<br />ally harder to attain, because seed size often prevents the<br />achievement of suitably rapid dehydration. However, not all<br />recalcitrant seeds are too large, or lose water too slowly, to<br />facilitate the achievement of very different drying rates. The<br />ability to achieve lower water contents while retaining viabili-<br />ty has been recorded for whole seeds of Avicennia marina<br />(Farrant and others 1985) and Quercus rubra (Pritchard 1991).<br />We have recently carried out studies to ascertain the effects of<br />drying rate on whole seeds of Ekebergia capensis, a tropical,<br />meliaceous, recalcitrant species) for which markedly different<br />drying rates can be achieved (Pammenter and others 1998).<br />The results obtained illustrated the effects of drying rate dra-<br />matically: viability loss was already apparent in slowly dried<br />seeds at high axis water contents [ 1.25 g water per g dry mate-<br />rial ( 55 percent, wmb)] while those that were dehydrated rap-<br />idly showed unimpaired vigor and full germinability at an axis<br />water content of 0.7 g g-1 (40 percent, wmb). Seeds dried at an<br />intermediate rate retained viability to the intermediate axis<br />water content level of c. 1.0 g g-1 (50 percent, wmb). Ultra-<br />structural observations suggested that different damaging<br />mechanisms bring about intracellular damage, depending on<br />the drying rate. Advanced degradation of membranes, partic-<br />ularly of the plastids, and an abnormality of the lipid bodies<br />occurred in axes from slowly dried seeds at water contents in<br />the region of 1.1 g g-1 (52 percent, wmb) when viability had<br />declined to 37 percent. The damage became steadily worse<br />with slow drying to lower water contents, until, at 0.6 g g-1 (37<br />percent, wmb), only fragments of intracellular components<br />remained. At a water content of 0.57 g g-1 (36 percent, wmb),<br />axes from rapidly dried seeds (viability 80 percent) showed lit-<br />tle signs of intracellular damage; it was only at considerably<br />lower axis water contents that signs of deterioration were<br />noted, which coincided with declining viability. At no stage<br />did the extensive degradation that characterized axis cells<br />from slowly dried seeds occur, supporting the proposal that if<br />desiccation-sensitive material can pass quickly enough through<br />water content ranges at which lethal reactions are prevalent,<br />then it is possible to dry the material down to a far lower<br />hydration level (see Vertucci and Farrant 1995 for discussion<br />of the various hydration levels).<br /> <br /><br /><br />146<br /> <br /><br /><br />Part I—Technical Chapters <br /><br /><br /><br /><br />There will be a water content at which rapidly dried<br />material that is desiccation sensitive will sustain injury, and,<br />while the value varies from species to species, it is usually near<br />the range where only structure-associated (nonfreezable)<br />water remains (Pammenter and others 1991, 1993; Pritchard<br />1991). Damage occurring at such relatively low water contents<br />is defined as desiccation damage in the strict sense (Pam-<br />menter and others 1998; Walters and others 2001) and is sug-<br />gested to coincide with the perturbation of the nonfreezable<br />water (Pammenter and others 1991). In contrast, desiccation-<br />tolerant material can withstand the removal of a considerable<br />proportion of this water (Pammenter and others 1991, Vertuc-<br />ci and Farrant 1995).<br />Slowly dried desiccation-sensitive material sustains dam-<br />age at relatively high water contents, certainly those where<br />solution (i.e. freezable) water prevails. This damage is suggest-<br />ed to result from aqueous-based, degradative reactions that are<br />the result of unbalanced metabolism (Pammenter and others<br />2001; Walters and others 2001). Recalcitrant seeds (and,<br />indeed, probably all nonorthodox types) are hydrated and<br />metabolically active when shed (Berjak and others 1989, Ber-<br />jak and Pammenter 1997). As water is slowly lost, metabolism<br />will continue, but when the seeds are still at relatively high<br />water contents, metabolism will become unbalanced or out-of-<br />phase as a result of internal water stresses (Senaratna and<br />McKersie 1986, Smith and Berjak 1995, Vertucci and Farrant<br />1995). A likely consequence of this unregulated metabolism<br />will be the generation of free radicals and accompanying<br />oxidative damage (Finch-Savage and others 1994a, Hendry<br />1993, Hendry and others 1992, Leprince and others 1990b).<br />The severity of this type of damage, which is being termed<br />metabolic damage (Walters and others 2001), is predicted to<br /> <br /><br /><br /><br /><br />increase in inverse proportion to the drying rate, with viabili-<br />ty loss occurring at increasingly high water contents.<br /><br /><br /><br /><br />CONCLUDING COMMENTS<br /><br /><br /><br />It is proposed that nonorthodox seed behavior is a consequence<br />of the lack of some, or perhaps all, of the suite of protective<br />mechanisms or processes that together confer desiccation tol-<br />erance on orthodox seeds. There is likely to be a gradation in<br />the presence and/or efficacy of the proposed processes/mech-<br />anisms among seeds of nonorthodox species, accounting for<br />the variability of the responses to stresses, particularly that<br />imposed by dehydration. The most desiccation-sensitive recal-<br />citrant seeds are probably those that lack virtually all the pro-<br />tective and restitutional factors that facilitate the acquisition<br />and maintenance of desiccation tolerance in orthodox seeds.<br />Two major factors are proposed to contribute to the loss<br />of viability of recalcitrant seeds: (1) the consequences of unbal-<br />anced metabolism during dehydration [and possibly also when<br />such seeds are stored in the hydrated condition (Smith and<br />Berjak 1995)]; (2) desiccation damage in the strict sense, which<br />occurs when water that is essential for the integrity of intracel-<br />lular structures is removed; in recalcitrant seeds, this equates<br />with nonfreezable water (Pammenter and others 1991).<br />We will probably be unable to account satisfactorily for<br />nonorthodox seed behavior, particularly that of truly recalci-<br />trant seeds, until complete understanding is gained of the<br />apparently numerous interacting factors that enable desicca-<br />tion-tolerance to be achieved.<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />Chapter 4: Orthodox and Recalcitrant SeedsUnknownnoreply@blogger.comtag:blogger.com,1999:blog-8393897017990891850.post-50320831604402938642010-08-31T04:10:00.000-07:002010-08-31T04:22:55.435-07:00PENYIMPANAN BENIHProgram studi agroteknologi<br /><br />I. PENDAHULUAN<br /><br />Selama ribuan tahun petani di seluruh dunia telah memproduksi dan menyimpan benih mereka sendiri. Disamping memproduksi makanan untuk keluarga mereka, para petani di seluruh dunia menyimpan benih benih dari tanaman mereka yang tersehat dan terbaik kualitasnya. Dengan meniru proses alami di sekitarnya, para penyimpan benih telah membentuk beranekaragam varietas berkwalitas seperti yang masih kita rasakan pada saat ini.<br />Penyimpanan benih merupakan salah satu cara yang dapat menunjang keberhasilan penyediaan benih, mengingat bahwa kebanyakan jenis pohon hutan tidak berbuah sepanjang tahun sehingga perlu dilakukan penyimpanan yang baik agar dapat menjaga kestabilan benih dari segi kuantitas maupun kualitasnya (Widodo, 1991).<br />Menurut Schmidt (2000), tujuan utama penyimpanan benih adalah untuk menjamin persediaan benih yang bermutu bagi suatu program penanaman bila diperlukan. Jika waktu penyemaian dilaksanakan segera setelah pengumpulan benih maka benih dapat langsung digunakan di persemian sehingga penyimpanan tidak diperlukan. Akan tetapi kasus semacam ini sangat jarang terjadi, hal ini disebabkan karena pada daerah dengan iklim musim yang memiliki musim penanaman pendek sangat tidak memungkinkan untuk langsung menyemai benih, sehingga benih perlu disimpan untuk menunggu saat yang tepat untuk disemai.<br /><br />II. ISI<br /><br />Penyimpanan dalam rangka pembenihan mempunyai arti yang luas, karena yang diartikan penyimpanan di sini adalah sejak benih itu mencapai kemasakan fisiologisnya sampai ditanam. Adapun tempat dan waktunya bisa terjadi ketika benih masih berada pada tanaman, di gudang penyimpanan atau dalam rangka pengiriman benih itu ke tempat atau daerah yang memerlukan. Selama dalam penyimpanan karena pengaruh beberapa faktor, mutu benih akan mengalami kemunduran Kartasapoetra(1986) dalam Hario Polije(2009) . Selama penyimpanan benih, proses fisiologis tetap berlangsung sehingga harus diusahakan agar proses ini berjalan seminimal mungkin Hendarto (1996) dalam Hario Polije(2009). Tujuan utama penyimpanan benih adalah untuk mempertahankan viabilitas benih selama periode simpan yang lama, sehingga benih ketika akan dikecambahkan masih mempunyai viabilitas yang tidak jauh berbeda dengan viabilitas awal sebelum benih disimpan.<br />Kegiatan penyimpanan benih tidak terlepas dari penggunaan wadah simpan. Menurut Siregar (2000) dalamYudi Harisman (2009), beberapa sifat khusus yang harus diperhatikan dari wadah simpan adalah :<br />1. Permeabilitas, yaitu kemampuan wadah untuk dapat menahan kelembaban dan gas pada level tertentu<br />2. Insulasi, yaitu kemampuan wadah untuk mempertahankan suhu<br />3. Ukuran lubang, yaitu kemampuan wadah untuk bertahan dari serangan serangga dan mikroorganisme yang dapat masuk melalui celah-celah kemasan<br />4. Kemudahan dalam hal penanganan seperti tidak licin, mudah ditumpuk, mudah dibuka, ditutup, disegel dan mudah dibersihkan.<br />5. Biaya, harus diperhitungkan dengan nilai nominal dari benih sendiri<br />Wadah simpan pada dasarnya dapat digolongkan menjadi 2 (dua) macam yakni wadah yang kedap udara dan wadah yang permeable Widodo(1991) dalam Yudi Harisman (2009). Wadah kedap adalah wadah yang tidak memungkinkan lagi terjadi pertukaran udara antara benih yang disimpan dengan lingkungannya, sedangkan wadah permeabel adalah wadah yang masih memungkinkan terjadinya pertukaran udara antara benih dengan lingkungannya.<br />Menurut Siregar (2000) dalam Yudi Harisman (2009)., contoh dari wadah yang permeabel adalah karung goni, kantong kain, karung nilon, keranjang, kotak kayu, kertas, karton dan papan serat yang tidak dilapisi lilin. Sedangkan wadah yang tidak permeabel adalah kaleng logam, botol dan gelas. Justice dan Bass (1979) dalam Yudi Harisman (2009)., mengemukakan bahwa penggunaan wadah dan cara simpan benih sangat tergantung pada jenis, jumlah benih, teknik pengepakan, lama penyimpanan, suhu ruang simpan dan kelembaban ruang simpan.<br />Berapa lama benih dapat disimpan sangat tergantung pada kondisi benih dan lingkungannya sendiri. Beberapa tipe benih tidak mempunyai ketahanan untuk disimpan dalam jangka waktu yang lama atau sering disebut benih rekalsitran. Sebaliknya benih ortodoks mempunyai daya simpan yang lama dan dalam kondisi penyimpanan yang sesuai dapat membentuk cadangan benih yang besar di tanah Schmidt (2000) dalam Yudi Harisman (2009).. Meskipun tipe ortodoks dan rekalsitran relatif jelas perbedaannya, daya tahan benih untuk bertahan pada saat penyimpanan meliputi variasi yang luas, dari yang sangat rekalsitran, intermediate sampai ortodoks. Pada umumnya semakin lama benih disimpan maka viabilitasnya akan semakin menurun. Mundurnya viabilitas benih merupakan proses yang berjalan bertingkat dan kumulatif akibat perubahan yang diberikan kepada benih mengemukakan bahwa periode penyimpanan terdiri dari penyimpanan jangka panjang, penyimpanan jangka menengah dan penyimpanan jangka pendek. Penyimpanan jangka panjang memiliki kisaran waktu puluhan tahun, sedangkan penyimpanan jangka menengah memiliki kisaran waktu beberapa tahun dan penyimpanan jangka pendek memiliki kisaran waktu kurang dari satu tahun. Tidak ada kisaran pasti dalam periode penyimpanan, hal ini disebabkan karena periode penyimpanan sangat tergantung dari jenis tanaman dan tipe benih itu sendiri.<br />Ketahanan benih untuk disimpan beragam tergantung dari jenis, cara dan tempat penyimpanan Sutopo (1988) dalamHario Polije(2009). Dalam kegiatan penanganan benih, secara umum benih dikelompokkan ke dalam dua golongan utama sesuai dengan kondisi penyimpanan yang dituntut, yaitu benih recalsitrant dan benih orthodox. Benih orthodox mampu disimpan dalam waktu yang lama pada kadar air benih yang rendah (2 – 5%) dan suhu penyimpanan yang rendah. Benih recalsitrant adalah benih yang viabilitasnya segera turun sampai nol jika disimpan dalam waktu yang lama dan kadar air yang rendah. Pada benih recalsitrant, kadar air benih pada waktu masak lebih dari 30% sampai 50%, dan sangat peka terhadap pengeringan di bawah 12% sampai 30%. Kelompok species yang benihnya tahan terhadap pengeringan sampai kadar air benih yang rendah seperti pada benih orthodox, tetapi sangat peka terhadap suhu penyimpanan yang rendah, belakangan ini dikelompokkan dalam benih intermediate (Ellis et al., 1990 dalam Schmidt, 2000).<br />Menurut Schmidt (2000) dalam Hario Polije (2009), benih orthodox tahan terhadap pengeringan dan suhu penyimpanan yang rendah, yaitu pada suhu 0 – 5o C dengan kadar air benih 5 – 7%. Dalam kondisi penyimpanan yang optimal, benih yang orthodox akan mampu disimpan sampai beberapa tahun. Pada saat masak, kadar air benih pada kebanyakan benih orthodox sekitar 6 – 10%. Benih orthodox banyak ditemukan pada zona arid, semi arid dan pada daerah dengan iklim basah, di samping itu juga ada yang ditemukan pada zona tropis dataran tinggi. Menurut Schmidt (2000), benih recalsitrant didefinisikan sebagai benih yang tidak tahan terhadap pengeringan dan suhu penyimpanan yang rendah, kecuali untuk beberapa species temperate recalsitrant. Tingkat toleransinya tergantung dari species masing-masing, umtuk benih species dari daerah tropik kadar air benih yang dianjurkan untuk penyimpanan adalah 20 – 35% dan suhu penyimpanan 12 – 15o C. kebanyakan benih recalsitrant hanya mampu disimpan beberapa hari sampai dengan beberapa bulan. Benih recalsitrant pada waktu masak, kadar air benih sekitar 30 – 70%. Benih recalsitrant banyak ditemukan pada species dari zona iklim tropis basah, hutan hujan tropis, dan hutan mangrove, beberapa ditemukan pada zona temperate dan sedikit ditemukan pada zona panas.<br />Benih yang diproduksi dan diproses seringkali tidak langsung ditanam tetapi disimpan dahulu untuk digunakan pada musim tanam berikutnya, di samping itu ada pula benih yang memang perlu disimpan dalam waktu tertentu terlebih dahulu sebelum ditanam yaitu benih yang mengalami after ripening. Untuk menghambat laju deteriorasi maka benih ini harus disimpan dengan metode tertentu agar benih tidak mengalami kerusakan ataupun penurunan mutu.<br />Kunci keberhasilan penyimpanan benih ortodoks seperti jagung terletak pada pengaturan kadar air dan suhu ruang simpan. Hal tersebut sesuai dengan hasil penelitian yang dikemukakan oleh Harrington (1972) danDelouche (1990) dalam M. Azrai (dkk.). Namun demikian, suhu hanya berperan nyata pada kondisi kadar air di mana sel-sel pada benih memiliki air aktif (water activity)yang memungkinkan proses metabolisme dapat berlangsung. Proses metabolisme meningkat dengan meningkatnya kadar air benih, dandipercepat dengan meningkatnya suhu ruang simpan. Peningkatan metabolisme benih menyebabkan kemunduran benih lebih cepat (Justiceand Bass 1979). Kaidah umum yang berlaku dalam penyimpanan benih menurut Matthes et al. (1969) adalah untuk setiap 1% penurunan kadar air,daya simpan dua kali lebih lama. Kaidah ini berlaku pada kisaran kadar air5-14%, dan suhu ruang simpan tidak lebih dari 40oC.<br />Secara praktis, benih dapat disimpan pada suhu kamar (28oC) atauruang sejuk (12oC), bergantung pada lama penyimpanan dan kadar air benihyang akan disimpan. Apabila daya berkecambah benih dipertahankan diatas 80% (sesuai standar daya berkecambah), maka kadar air benih harus12% (dapat dicapai melalui pengeringan dengan sinar matahari pada musimkemarau) agar daya berkecambah benih masih dapat dipertahankansampai 10 bulan penyimpanan pada suhu kamar (28oC). Kalau kadar airbenih dapat diturunkan hingga 10%, daya berkecambah benih dapatdipertahankan sampai 14 bulan, dan lebih dari 14 bulan kalau kadar airbenih pada saat disimpan 8%. Daya berkecambah benih setelahpenyimpanan 14 bulan masih tinggi (89,3%). Di lain pihak, pada kadar air14%, benih hanya tahan disimpan selama delapan bulan, dan pada kadarair 16% hanya tahan disimpan sampai empat bulan. (M. Azrai, dkk)<br />Penyimpanan pada suhu sejuk (12oC), daya berkecambah benih masih di atas 80% dengan kadar air 16% dan dapat bertahan selama enam bulan. Apabila kadar air diturunkan menjadi 14%, benih akan bertahan sampai 12bulan dan pada kadar air 8-12% dapat bertahan sampai 18 bulan. Daya simpan benih selain bergantung pada suhu ruang simpanjuga bergantung pada kadar air awal. Jika disimpan pada kadar air <10%pada>oC, daya berkecambah masih di atas 80% sampaipada penyimpanan 16 bulan. Jika kadar air dinaikkan menjadi 12%, dayaberkecambah benih pada penyimpanan 16 bulan hanya sekitar 60%, padakadar air 14% daya berkecambahnya hanya 40%, bahkan pada kadar 16%benih sudah tidak berkecambah setelah penyimpanan enam bulan. (M.Azrai, dkk)<br /><br /><br />III. PENUTUP<br /><br />Sebagai penutup dalam makalah ini bahwapenyimpanan benih merupakan salah satu cara yang dapat menunjang keberhasilan penyediaan benih. Penyimpanan ini mempunyai tujuan utama penyimpanan benih adalah untuk menjamin persediaan benih yang bermutu bagi suatu program penanaman bila diperlukan dan untuk mempertahankan viabilitas benih selama periode simpan yang lama, sehingga benih ketika akan dikecambahkan masih mempunyai viabilitas yang tidak jauh berbeda dengan viabilitas awal sebelum benih disimpan.<br />Penyimpanan pada suhu sejuk (12oC), daya berkecambah benih masihdi atas 80% dengan kadar air 16% dan dapat bertahan selama enam bulan.Apabila kadar air diturunkan menjadi 14%, benih akan bertahan sampai 12bulan dan pada kadar air 8-12% dapat bertahan sampai 18 bulan Namun demikian, suhu hanya berperan nyata pada kondisi kadar air di mana sel-sel pada benih memiliki air aktif (water activity)yang memungkinkan proses metabolisme dapat berlangsung. benih dapat disimpan pada suhu kamar (28oC) atauruang sejuk (12oC), bergantung pada lama penyimpanan dan kadar air benihyang akan disimpanApabila daya berkecambah benih dipertahankan diatas 80% (sesuai standar daya berkecambah), maka kadar air benih harus12% (dapat dicapai melalui pengeringan dengan sinar matahari pada musimkemarau) agar daya berkecambah benih masih dapat dipertahankansampai 10 bulan penyimpanan pada suhu kamar (28oC)<br />keberhasilan penyimpanan benih ortodoks seperti jagung terletak pada pengaturan kadar air dan suhu ruang simpan<br /><br /><br />DAFTAR PUSTAKA<br />M. Azrai, Rahmawati, Ramlah Arief dan Sania Saenong. Pengelolaan Benih Jagung. Balai Penelitian Tanaman Serealia, Maros.http://balitsereal.litbang.deptan.go.id/ind/bjagung/sebelas.pdf diakses pada tanggal 9 Juni 2010.<br />Hendarto(1996), Kartasapoetra(1986), Schmidt (2000), Sutopo(1988) dalam Hario Polije. 2009. Penyimpanan benih (seed storage).http://hariopolije.blogspot.com/2009/04/hmmm.html. diakses pada tanggal 9 Juni 2010.<br />Justice and Bass(1979), Schmidt, L(2000), Siregar, S.T(2000), Widodo, W (1991) dalam Yudi Harisman, 2009. Wadah dan Lama Penyimpanan Benih. http://forester-rimbawan.blogspot.com/2009/05/wadah-dan-lama-penyimpanan-benih.html diakses pada tanggal 9 Juni 2010.<br />Yayasan IDEP, Lembaran Fakta Yayasan Idep Tentang Penyimpanan Benih Dan Perkembangbiakan Tanaman.http://www.idepfoundation.org/indonesia/ download_files/ seed_ saving/Fsheet_seeds_indo.pdf diakses pada tanggal 9 Juni 2010.Unknownnoreply@blogger.comtag:blogger.com,1999:blog-8393897017990891850.post-34391192901171437752010-05-26T08:42:00.000-07:002010-05-26T09:35:09.255-07:00Head Tail Game Liberty ReserveMungkin sudah banyak yang mengetahui tentang permainan ini, dengan bermacam-macam strategi atupun trik-trik yang mereka sarankan. Tips ini hanya berlaku untuk permainan yang memberikan keuntungan 200% atau lebih, sehingga bisa dikatakan dalam permainan ini kita pasti bisa mengambil keuntungan atau 99% win. Sebenarnya permainan ini tidak jauh beda dengan permainan tebak-tebakan sisi koin mana yang akan keluar selanjutnya, dalam hal ini kemungkinannya adalah 50-50.<br />Mengapa saya anjurkan LibertyReserve game?<br />Salah satu alasan kenapa saya menganjurkan Liberty Reserve game karena pembayaran atas kemenangan kita akan langsung masuk ke acount Liberty Reserve begitu permainan selesai. Jadi kita bisa menikmati hasil dari permainan kita langsung tanpa harus menunggu terlalu lama.<br /><br /><span style="font-weight:bold;">Apa itu LibertyReserve?</span><br /><br />LibertyReserve, Ltd adalah suatu perusahaan finansial ternama di dunia yang berpusat di Amerika, yang mengkhususkan dirinya pada transaksi internet. Bisa dikatakan bahwa secara de-facto LibertyReserve adalah mata uang internet. Transaksi dengan menggunakan kartu kredit sangat rawan terhadap pembobolan kartu kredit. Karena itu, LibertyReserve menawarkan solusi pembayaran di internet yang relatif lebih aman. Cara kerja LibertyReserve persis seperti bank-bank tradisional pada umumnya. LibertyReserve menerima deposit dari para nasabahnya, dimana nasabahnya dapat bertransaksi transfer keluar ataupun masuk melalui LibertyReserve. Persis seperti bank tradisional pada umumnya.<br />Bagaimana cara mendapatkan account LibertyReserve ???<br />Mudah sekali, cukup hanya dengan mengisikan aplikasi pendaftaran secara gratis pada website <span style="font-weight:bold;"><a href="http://www.libertyreserve.com/?ref=U1588482">www.LibertyReserve.com</a></span>, anda sudah dapat mendapatkan account LibertyReserve secara gratis.<br />Account LibertyReserve anda baru berguna apabila anda memiliki uang di dalamnya. Sama seperti account anda di bank tradisional. Apabila tidak memiliki dana, jangan harap dapat transfer ke luar, walaupun hanya $0,1. Untuk membeli LibertyReserve anda dapat mengunjungi <a href="http://www.sentraegold.com/?sentra=89071">http://www.sentraegold.com/</a> atau <a href="http://www.greatachiever.com/index.html?ref=4224">http://www.greatachiever.com/</a>, sebuah toko LibertyReserve online yang menerima pembelian maupun penjualan LibertyReserve di Indonesia. Apabila lokasi anda di Indonesia, anda bisa mencari website yang menjual LibertyReserve dengan rupiah di google dengan keyword “jual beli LibertyReserve”.<br />Berikut tips bermain LibertyReserve (LR) Game Head and Tail 99% Win<br /><span style="font-weight:bold;">• Pilih salah satu situs LR game rekomendasi dibawah ini untuk memulai bermain:</span> <br />o <a href="http://www.bettergold4u.com/lr_headtail.php?refU1588482">www.bettergold4u.com</a><br />o <a href="http://www.1gold-game.com/liberty_headtail/index.php?ref=U1588482">www.1gold-game.com</a><br />o <a href="http://www.luckygold.info/?ref=U1588482">www.luckygold.info</a><br />o <a href="http://www.gugold.com/index.php?ref=U1588482">www.gugold.com</a><br />o <a href="http://www.1goldgame.com/index.php?ref=U1588482">www.1goldgame.com</a><br /><span style="font-weight:bold;">• Pilih taruhan dari yang terkecil dulu.</span><br /><span style="font-weight:bold;">• Pilih “head atau tail” terserah anda. Di games lain seperti left & right, low & high, red & black, dan yang lainnya, berlaku sama seperti head & tail, cuma beda nama saja!</span><br /><span style="font-weight:bold;">• Klik “Play”</span><br />Bayar bet-nya dengan libertyreserve<br />Setelah itu anda akan tahu menang atau kalah.<br />Jika menang, ulangi langkah ke-1<br />Jika kalah, bet lagi dengan jumlah dobel dari kekalahan. Gunakan pilihan sama dengan kekalahan sebelumnya.<br />Misal tadi kalah $1 dengan pilihan “tail”, maka sekarang bet $2 harus “tail”!<br />Dengan begitu, jika menang, anda tetap untung meski tadi sebelumnya kalah!<br />Hitungannya jika menang, anda dapat 2x (bet $2 dapat $4, untung $2 dikurangi $1 (kekalahan sebelumnya) anda masih untung $1 !!!)<br /><span style="font-weight:bold;">• Selanjutnya anda bisa mengulang-ulang langkah diatas</span><br />Pertahankan pilihan yang sama itu sampai anda memperoleh kemenangan! Ingat setiap kali kalah, anda harus bet 2x lipat dari kekalahan sebelumnya.<br />Anda bisa stop jika dirasa sudah cukup. <br /><br />Ini detail dari langkah-langkah betting: (contoh "Head")<br />Langkah Pilihan Bet Win 250% Total Lost <br /><br /><span style="font-weight:bold;">1st Head $0.5 $1.25 $0.5<br />2nd Head $1 $2.5 $1.5<br />3rd Head $2 $5 $3.5<br />4th Head $4 $10 $7.5<br />5th Head $8 $20 $15.5<br />6th Head $16 $40 $31.5<br />7th Head $32 $80 $63.5</span><br /><br />Contoh hitungannya begini:<br />Kalah 5x berturut-turut = $0,5+$1+$2+$4+$8 = $15,5<br />Lalu bet selanjutnya anda adalah $16(dobel/ganda dari kekalahan sebelumnya yaitu $8). Jika menang anda dapat = $40 (250% x $16 = $40)<br />Maka anda tetap untung = $40 - modal $16 - $15,5 =$8,5<br />Itu hitungan sebenarnya<br />Jadi setiap kali anda menang, anda untung $8,5 bahkan bisa lebih besar, tidak peduli berapa banyak anda kalah karena setiap 1x menang, kekalahan sebelumnya akan lunas oleh "hanya" sekali menang.<br />Jadi, anda bisa memilih “head” & “tail” 7x berturut-turut. Tapi sepengalaman saya, paling tinggi hanya 5x kalah berturut-turut!”<br />“Perhatian!” 100% Win berlaku jika anda sudah menjalankan maksimal 10 langkah tersebut. Dibawah 10 langkah, berdasarkan pengalaman, saya hanya pernah kalah paling banyak sampai Langkah ke-5, tapi menang di Langkah ke-6. Jadi belum pernah kalah lebih dari Langkah 6“<br /><br />Selamat mencoba, Good Luck!<br />Nb : Apapun juga, ini hanya permainan kalah atau menang hal yang biasa.Yang penting jangan penasaran / nafsu, jika menang atau kalah.Unknownnoreply@blogger.comtag:blogger.com,1999:blog-8393897017990891850.post-91338438067145499632010-05-23T07:48:00.001-07:002010-05-23T07:48:55.817-07:00Produktivitas Kerja : Definisi dan Pengukuran Produktivitas Tenaga KerjaProduktivitas merupakan nisbah atau rasio antara hasil kegiatan (output, keluaran) dan segala pengorbanan (biaya) untuk mewujudkan hasil tersebut (input, masukan) (Kussriyanto, 1984, p.1). Input bisa mencakup biaya produksi (production cost) dan biaya peralatan (equipment cost). Sedangkan output bisa terdiri dari penjualan (sales), earnings (pendapatan), market share, dan kerusakan (defects) (Gomes,1995, p.157).<br />Produktivitas tenaga kerja adalah salah satu ukuran perusahaan dalam mencapai tujuannya. Sumber daya manusia merupakan elemen yang paling strategik dalam organisasi, harus diakui dan diterima oleh manajemen. Peningkatan produktivitas kerja hanya mungkin dilakukan oleh manusia (Siagian, 2002, p.2). Oleh karena itu tenaga kerja merupakan faktor penting dalam mengukur produktivitas. Hal ini disebabkan oleh dua hal, antara lain; pertama, karena besarnya biaya yang dikorbankan untuk tenaga kerja sebagai bagian dari biaya yang terbesar untuk pengadaan produk atau jasa; kedua, karena masukan pada faktor-faktor lain seperti modal (Kussriyanto, 1993, p.1).<br />Menurut Anoraga dan Suyati, (1995, p.119-121) produktivitas mengandung pengertian yang berkenaan dengan konsep ekonomis, filosofis dan sistem. Sebagai konsep ekonomis, produktivitas berkenaan dengan usaha atau kegiatan manusia untuk menghasilkan barang atau jasa yang berguna untuk pemenuhan kebutuhan manusia dan masyarakat pada umumnya.<br />Sebagai konsep filosofis, produktivitas mengandung pandangan hidup dan sikap mental yang selalu berusaha untuk meningkatkan mutu kehidupan dimana keadaan hari ini harus lebih baik dari hari kemarin, dan mutu kehidupan hari esok harus lebih baik dari hari ini. Hal inilah yang memberi dorongan untuk berusaha dan mengembangkan diri. Sedangkan konsep sistem, memberikan pedoman pemikiran bahwa pencapaian suatu tujuan harus ada kerja sama atau keterpaduan dari unsur-unsur yang relevan sebagai sistem.<br />Dapat dikatakan bahwa produktivitas adalah perbandingan antara hasil dari suatu pekerjaan karyawan dengan pengorbanan yang telah dikeluarkan. Hal ini sesuai dengan pendapat Sondang P. Siagian bahwa produktivitas adalah: “Kemampuan memperoleh manfaat yang sebesar-besarnya dari sarana dan prasarana yang tersedia dengan menghasilkan output yang optimal bahkan kalau mungkin yang maksimal.”<br />Banyak hasil penelitian yang memperlihatkan bahwa produktivitas sangat dipengaruhi oleh faktor: knowledge, skills, abilities, attitudes, dan behaviours dari para pekerja yang ada di dalam organisasi sehingga banyak program perbaikan produktivitas meletakkan hal-hal tersebut sebagai asumsi-asumsi dasarnya (Gomes, 1995, p.160).<br /><br />Pengertian lain dari produktivitas adalah suatu konsep universal yang menciptakan lebih banyak barang dan jasa bagi kehidupan manusia, dengan menggunakan sumber daya yang serba terbatas (Tarwaka, Bakri, dan Sudiajeng, 2004, p.137).<br />Menurut Manuaba (1992) peningkatan produktivitas dapat dicapai dengan menekan sekecil-kecilnya segala macam biaya termasuk dalam memanfaatkan sumber daya manusia (do the right thing) dan meningkatkan keluaran sebesar-besarnya (do the thing right). Dengan kata lain bahwa produktivitas merupakan pencerminan dari tingkat efisiensi dan efektivitas kerja secara total (Tarwaka, Bakri, dan Sudiajeng, 2004, p.138).<br />Menurut Sinungan, (2003, p.12), secara umum produktivitas diartikan sebagai hubungan antara hasil nyata maupun fisik (barang-barang atau jasa) dengan masuknya yang sebenarnya. Produktivitas juga diartikan sebagai tingkatan efisiensi dalam memproduksi barang-barang atau jasa-jasa. Produktivitas juga diartikan sebagai:<br />a. Perbandingan ukuran harga bagi masukan dan hasil<br />b. Perbedaan antara kumpulan jumlah pengeluaran dan masukan yang dinyatakan dalam satuan-satuan (unit) umum.<br />Ukuran produktivitas yang paling terkenal berkaitan dengan tenaga kerja yang dapat dihitung dengan membagi pengeluaran oleh jumlah yang digunakan atau jam-jam kerja orang.<br /><br />Pengukuran Produktivitas Tenaga Kerja<br />Pengukuran produktivitas tenaga kerja menurut system pemasukan fisik perorangan/perorang atau per jam kerja orang diterima secara luas, namun dari sudut pandangan/ pengawasan harian, pengukuran-pengukuran tersebut pada umumnya tidak memuaskan, dikarenakan adanya variasi dalam jumlah yang diperlukan untuk memproduksi satu unit produk yang berbeda. Oleh karena itu, digunakan metode pengukuran waktu tenaga kerja (jam, hari atau tahun). Pengeluaran diubah ke dalam unit-unit pekerja yang biasanya diartikan sebagai jumlah kerja yang dapat dilakukan dalam satu jam oleh pekerja yang terpercaya yang bekerja menurut pelaksanaan standar.<br />Karena hasil maupun masukan dapat dinyatakan dalam waktu, produktivitas tenaga kerja dapat dinyatakan sebagai suatu indeks yang sangat sederhana = Hasil dalam jam-jam yang standar : Masukan dalam jam-jam waktu.<br />Untuk mengukur suatu produktivitas perusahaan dapatlah digunakan dua jenis ukuran jam kerja manusia, yakni jam-jam kerja yang harus dibayar dan jam-jam kerja yang dipergunakan untuk bekerja. Jam kerja yang harus dibayar meliputi semua jam-jam kerja yang harus dibayar, ditambah jam-jam yang tidak digunakan untuk bekerja namun harus dibayar, liburan, cuti, libur karena sakit, tugas luar dan sisa lainnya. Jadi bagi keperluan pengukuran umum produktivitas tenaga kerja kita memiliki unit-unit yang diperlukan, yakni: kuantitas hasil dan kuantitas penggunaan masukan tenaga kerja (Sinungan, 2003, p.24-25).<br /><br />Menurut Wignjosoebroto, (2000, p.25), produktivitas secara umum akan dapat diformulasikan sebagai berikut:<br />Produktivitas = Output/input(measurable)+ input (invisible).<br />Invisible input meliputi tingkat pengetahuan, kemampuan teknis, metodologi kerja dan pengaturan organisasi, dan motivasi kerja.<br />Untuk mengukur produktivitas kerja dari tenaga kerja manusia, operator mesin, misalnya, maka formulasi berikut bisa dipakai untuk maksud ini, yaitu:<br />Produktivitas = total keluaran yang dihasilkan<br />Tenaga Kerja jumlah tenaga kerja yang dipekerjakan Di sini produktivitas dari tenaga kerja ditunjukkan sebagai rasio dari jumlah keluaran yang dihasilkan per total tenaga kerja yang jam manusia (man-hours), yaitu jam kerja yang dipakai untuk menyelesaikan pekerjaan tersebut. Tenaga kerja yang dipekerjakan dapat terdiri dari tenaga kerja langsung ataupun tidak langsung, akan tetapi biasanya meliputi keduanya.<br />Labels: Manajemen Sumber Daya ManusiaUnknownnoreply@blogger.com