Antimicrobial forcing solution improves recovery of cryopreserved temperate fruit tree dormant buds

Dormant bud cryogenic preservation is a cost- and labor-efficient method of genetic resource backup compared to in vitro derived meristem shoots cryopreservation. While protocols have been developed for cryopreserving apple dormant buds, effective and reproducible protocols are yet to be developed for several temperate fruit and nut species. Dormant bud cryopreservation typically requires material to be grafted to evaluate viability and recover a plant. Forced bud development has been used on a very limited scale for cryostored dormant budwood recovery, however, it provides a labor-efficient alternative viability assessment. To increase the utility of this approach, regrowth must be optimized to allow complete plant recovery. We hypothesized that bacterial attacks are limiting regrowth, thus, an antimicrobial forcing solution can maximize regrowth potential. This study examined the effects of an antimicrobial forcing solution (8-hydroxyquinoline citrate and sucrose, 8-HQC) on the cryosurvival and recovery of dormant buds of fruit (Malus x domestica, Prunus armeniaca, Prunus avium, Prunus persica, Pyrus communis), and nut species (Juglans regia, Juglans nigra, Juglans microcarpa). Recovery and shoot development were significantly improved for all the fruit and one nut species (J. microcarpa) treated with the 8-HQC, compared to standard recovery under high humidity alone (P < 0.001). Additionally, this post cryo recovery approach led to successful in vitro shoot tip establishment across all surviving fruit species. 8-HQC embedded forced bud development method increased viability and efficiency for existing cryostored material and can be used as a benchmark to develop protocols for different crops that could potentially lead to complete plant recovery.     

Tiller hierarchy and defoliation frequency determine bud viability in the grass <Emphasis Type="Italic">Poa ligularis</Emphasis>

Bud viability after various defoliation frequency treatments was determined in the perennial bunchgrass Poa ligularis under arid field conditions from 2002 to 2005. Bud respiratory activity was examined on various stem base hierarchies using the tetrazolium test, as validated with the vital stain Evan’s blue. The hypothesis of this work was that the total and viable axillary bud numbers on stem bases of all study stem base hierarchies are reduced as defoliation frequency increases. Interpretation of the results differed when they were expressed as a percentage rather than on a number per stem base basis. The total number of axillary buds per stem base was similar in all defoliation frequencies. When the results were expressed on a percentage basis, the order on stem bases having metabolically active buds was daughter tillers > stem bases with green tillers > stem bases without green tillers in all defoliation frequencies. The reverse order was found when considering dead buds. How the results are expressed thus deserves our attention when reporting results on bud viability in perennial grasses. An increased defoliation frequency increased the percentage of dead and dormant buds after the third or fourth defoliation of P. ligularis during the 1st study year. These percentages of bud viability, however, increased after the first defoliation during the 2nd study year. Bud viability was affected not only by the cumulative effects of defoliation but also by climatic variables throughout the seasons. However, our results show that P. ligularis can be defoliated up to twice a year without affecting bud viability, and thus its potential capacity for regrowth after defoliation.     

Droplet-vitrification methods for apical bud cryopreservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.) H. Rob.]

This study aimed to develop a cryopreservation protocol for the long-term preservation of yacon [Smallanthus sonchifolius (Poepp. and Endl.)], an Andean crop with high fructooligosaccharide content in its tuberous roots. Initially, the cryopreservation protocol was developed using a yacon clone originated from Ecuador classified as ECU 41. Osmotic dehydration of apical buds (2–3 mm long) was carried out by assessing two plant vitrification solutions, PVS2 (15, 30, and 60 min) at 0 °C and PVS3 (30, 45, 60, and 75 min) at 22 °C. After cryopreservation, the apical buds were thawed and placed on MS medium?±?0.1 mg l^?1 N⁶-benzyladenine (BA). The survival rates ranged from 37 to 90% within all treatments, with those subjected to PVS2 and PVS3 for 60 min showing the highest survival rates on MS medium without BA (87 and 90%, respectively). At 12 weeks post cryopreservation, these treatments also provided the highest regrowth rates, both reaching 73% of normally growing (shooting, rooting) plantlets. Survival rates on MS?+?0.1 mg l^?1 BA regrowth medium reached up to 90%; however, regrowth into normally rooted plantlets did not exceed 67% post cryopreservation. The optimized protocols were then applied to 4 additional yacon clones originated from Bolivia and Peru, classified as BOL 22, BOL 23, PER 12, and PER 14. This resulted in survival and regeneration rates ranging between 79.7–94.1% and 66.3–75.4% respectively. Our study shows that optimal cryopreservation protocols for the long-term conservation of yacon can be based on both PVS2 and PVS3 vitrification solutions.

     

Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis

This paper presents the advances made over the last decade in cryopreservation of economically important vegetatively propagated fruit trees. Cryopreservation protocols have been established using both dormant buds sampled on field-grown plants and shoot tips sampled on in vitro plantlets. In the case of dormant buds, scions are partially dehydrated by storage at − 5 °C, and then cooled slowly to − 30 °C using low cooling rates (c.a. 1 °C/h) before immersion in liquid nitrogen. After slow rewarming and rehydration of samples, regrowth takes place either through grafting of buds on rootstocks or excision of apices and inoculation in vitro. In the case of shoot tips of in vitro plantlets, the cryopreservation techniques employed are the following: controlled rate cooling procedures involving slow prefreezing followed by immersion in liquid nitrogen or vitrification-based procedures including encapsulation–dehydration, vitrification, encapsulation–vitrification and droplet-vitrification. The current status of cryopreservation for a series of fruit tree species including Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis is presented. Routine application of cryopreservation for long-term germplasm storage in genebanks is currently limited to apple and pear, for which large cryopreserved collections have been established at NCGRP, Fort Collins (USA), using dormant buds and in vitro shoot tips, respectively. However, there are a growing number of examples of pilot scale testing experiments under way for different species in various countries. Progress in the further development and application of cryopreservation techniques will be made through a better understanding of the mechanisms involved in the induction of tolerance to dehydration and cryopreservation in frozen explants.     

Cryopreservation by slow cooling of rat neuronal cells

Although primary neuronal cells are routinely used for neuroscience research, with potential clinical applications such as neuronal transplantation and tissue engineering, a gold standard protocol for preservation has not been yet developed. In the present work, a slow cooling methodology without ice seeding was studied and optimized for cryopreservation of rat cerebellar granular cells. Parameters such as cooling rate, plunge temperature and cryoprotective agent concentration were assessed using a custom built device based on Pye's freezer idea. Cryopreservation outcome was evaluated by post thawing cell viability/viable cell yield and in culture viability over a period of 14 days. The best outcome was achieved when 10% of Me₂SO as cryoprotective agent, a cooling rate of 3.1 ± 0.2 °C/min and a plunge temperature of −48.2 ± 1.5 °C were applied. The granular cells cryopreserved under these conditions exhibited a cell viability of 82.7 ± 2.7% and a viable cell yield of 28.6 ± 2.2%. Moreover, cell viability in culture remained above 50%, very similar to not cryopreserved cells (control). Our results also suggest that post-thaw viability (based on membrane integrity assays) not necessarily reflects the quality of the cryopreservation procedure and proper functionality tests must be carried out in order to optimize both post thaw viability/cell yield and in culture performance.     

Branch construction and bud defence status at the canopy surface of a West African rainforest

Branch specimens were collected from the very tops of tropical tree crowns in southern Cameroon, West Africa. An analysis of branching patterns revealed a consistency of form across unrelated taxa. All specimens showed evidence of rhythmic growth cither due to the regular occurrence of dormant terminal buds or due to sympodial growth with loss or flowering of terminal buds. Study of bud anatomy revealed an extensive array of protective devices associated with drought tolerance and herbivore defence. Normally a considerable excess of dormant, well protected axillary buds were present which (almost without exception) existed in a viable state. In very many instances the large dormant bud population was due to the presence of accessory buds, i.e. > 1 bud in the axil of each leaf. The microclimate at the outer surface of a tropical rainforest may experience a daily increase in temperature and associated depression in humidity; the canopy surface characteristics are more akin to chaparral shrub vegetation than to familiar rainforest understorey vegetation.     

Properties of peach flower buds which facilitate supercooling

Water in dormant peach (Prunus persica [L.] Batsch. var. `Harbrite') flower buds deep supercooled. Both supercooling and the freezing of water within the bud axis and primordium as distinct components depended on the viability of the bud axis tissue. The viability of the primordium was not critical. Supercooling was prevented by wounding buds with a dissecting needle, indicating that bud structural features were important. Bud morphological features appeared to prevent the propagation of ice through the vascular tissue and into the primordium. In dormant buds, procambial cells had not yet differentiated into xylem vessel elements. Xylem continuity between the bud primordium and adjacent tissues did not appear to be established until buds had deacclimated. It was concluded that structural, morphological, and physiological features of the bud facilitated supercooling.     

Influence of Preculture Variables on Microspore Embryo Production in Brassica napus ssp. oleifera cv. Duplo

The effect of four preculture variables on microspore embryoinduction and growth were examined: (1) the source of the budselected for culture (apical or axillary inflorescence; (2)the method of harvest (single harvest of whole inflorescenceor sequential harvest of individual buds; (3) the length ofthe bud (2, 3 or 4 mm); and (4) the application of a 4 ^°Cpretreatment to the bud after harvest. Microscopic and macroscopicanalysis of every anther used for culture permitted an assessmentof the following parameters: (1) the percentage of induced buds;(2) the number of induced anthers per induced bud; (3) the numberof productive buds (with macroscopic embryos) as a percentageof the induced buds; (4) the degree of induction per inducedanther (an estimate of the number of microspores in which initialembryogenic divisions had commenced); and (5) embryoid survival(the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of inducedanthers and all five parameters were components of the finalyield - the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67·0per cent) occurred with 2 mm sequentially harvested non-pretreatedbuds. Overall, the values decreased with increasing bud lengthand were lower for pretreated and axillary buds. In contrast,the two other estimates of induction - number of induced anthersper induced bud and degree of induction per induced anther -both had maximum values from 3 mm sequentially harvested, pretreatedbuds from apical inflorescences. The highest final yield ofembryos per cultured bud (44·9) was found with 2 mm non-pretreatedbuds taken from a single harvest of the apical inflorescence.The study therefore confirmed that the different componentsof the final embryo yield are differentially affected by thefour preculture variables tested. These variables must be controlledif reproducible results are to be achieved. Brassica napus, tape, anther culture, pollen, microspore, haploid     

Cryopreservation of fruit germplasm

Most temperate fruit species are genetically heterozygous and vegetatively propagated. Active collections of fruit genetic resources in Germany are generally maintained in the field, e.g., as potted plants for Fragaria and as trees for pome and stone fruit species. The plant material in active collections should be kept in duplicate to ensure security in case of disease or environmental disaster. The aim of this study was to develop an efficient complementary conservation strategy for fruit genetic resources. Although costly, fruit tree cultivars can be duplicated as field collections at a second site within the framework of the German Fruit Genebank, which is a decentralized fruit-specific network. Wild species accessions, particularly those of the genera Malus spp. (apple) and Fragaria spp. (strawberry) as well as strawberry cultivars, can also be duplicated by means of cryopreservation. In the current study, long-term cryopreservation was initiated for 194 Fragaria genotypes. A protocol combining vitrification with cold acclimation was effective and highly reproducible, with an average regrowth rate of 86%. In Malus, a general cryopreservation protocol based on dormant winter buds was adopted. Based on the results provided in this study, a combination of traditional ex situ conservation and cryopreservation can greatly improve the stability and security of fruit germplasm conservation.     

Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

The freezing behavior of dormant buds in larch, especially at the cellular level, was examined by a Cryo-SEM. The dormant buds exhibited typical extraorgan freezing. Extracellular ice crystals accumulated only in basal areas of scales and beneath crown tissues, areas in which only these living cells had thick walls unlike other tissue cells. By slow cooling (5 °C/day) of dormant buds to −50 °C, all living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation detectable by Cryo-SEM. However, the recrystallization experiment of these slowly cooled tissue cells, which was done by further freezing of slowly cooled buds with LN and then rewarming to −20 °C, confirmed that some of the cells in the leaf primordia, shoot primordia and apical meristem, areas in which cells had thin walls and in which no extracellular ice accumulated, lost freezable water with slow cooling to −30 °C, indicating ability of these cells to adapt by extracellular freezing, whereas other cells in these tissues retained freezable water with slow cooling even to −50 °C, indicating adaptation of these cells by deep supercooling. On the other hand, all cells in crown tissues and in basal areas of scales, areas in which cells had thick walls and in which large masses of ice accumulated, had the ability to adapt by extracellular freezing. It is thought that the presence of two types of cells exhibiting different freezing adaptation abilities within a bud tissue is quite unique and may reflect sophisticated freezing adaptation mechanisms in dormant buds.     

Modelling compensatory regrowth with bud dormancy and gradual activation of buds

Many plants show compensatory regrowth after herbivory and dormant buds often have an important role in compensatory responses. Theoretical models have shown that herbivore damage may select for a bud bank, i.e., a pool of dormant buds that are protected from herbivory and that are activated after herbivore damage. Earlier models assumed that undamaged plants cannot activate their dormant buds without damage, although they apparently have sufficient resources for successful seed production through the additional shoots dormant buds could produce. However, many plants are able to gradually activate buds over an extended period of time without any cue from damage. The aim of this study was to analyze how herbivory imposes selection for gradual mobilization of the bud bank. I assume that selection pressures that affect the fraction of buds active at each time point include damage by herbivores, time left to the end of season, and the opportunity costs of dormant buds. I modelled bud dynamics with gradual activation when there is a single damage event and (i) when the seed set of a shoot is not dependent on the time it is active, or (ii) when the seed set of a shoot diminishes with later activation. In addition, I analyzed how (iii) risk of repeated herbivory affects selection for gradual activation. Under these models, gradual activation is optimal over a wide range of herbivory pressures. Selection appears to favour activation of all buds at the beginning of the season only when herbivore pressure is weak and when early shoots have a higher seed set than late shoots. Alternatively, strong herbivore pressure and late damage may select for a large bud bank throughout the growing season, without gradual activation; the bud bank is only mobilized after damage. In this case, damaged plants can overcompensate, i.e. they have a higher seed set than undamaged plants with the same bud activation pattern. Selection for overcompensation demands a stronger herbivore pressure in this current model than in earlier bud bank models. The model never predicts selection for overcompensation when there is a risk of repeated herbivory. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low temperature exotherms of winter buds of hardy conifers

Differential thermal analysis (DTA) of winter buds and the excisedprimordial shoots of sub-alpine or sub-cold firs revealed thatthese buds had all low temperature exotherms around –30?C.However, no low temperature exotherm below –15?C was detectedin the spring buds. In the winter bud of Abies firma, a temperatefir native to Japan, a low temperature exotherm was detectedaround –20?C, which is higher by 10?C than that of sub-alpineor sub-cold firs. The low temperature exotherms of these firsoccurred at nearly the same temperatures that result in thedeath of these primordial shoots. On the other hand, littleor no low temperature exotherm was detected in the winter budsof sub-cold spruces. In larch winter buds, numerous small exothermswere observed, which are probably due to the many leaf primordiain the buds. Unlike many temperate deciduous broad-leaved trees,no low temperature exotherm was detected below –15?C inwinter twig xylem of conifers such as Abies, Picea, Pinus, Larixand Pseudotsuga. Thus, very hardy coniferous twigs can tolerateextracellular freezing to –70?C. ¹ Contribution No. 1907 from the Institute of Low TemperatureScience. (Received June 8, 1978; )     

A scanning electron microscope study of healthy pecan tissues showing the presence or absence of internal fungal contamination

Summary Healthy pecan, Caryaillinoensis (Wang) Koch, tissue was obtained from an 8-year-old grafted Cherokee tree. Dormant buds were gathered and stored until spring growth. After rigorous surface sterilization, halves of both stored and freshly harvested dormant buds and of actively growing shoots were plated onto sterile PDA. Corresponding halves wre fixed in FAA and processed for scanning electron microscopy (SEM). All plated dormant buds (both stored and freshly harvested) showed presence internally of a fungus, and SEM studies revealed hyphalike strands similar to those of the isolated fungi within cells of those buds. The spring flush was sterile in culture, and strands were absent in SEM studies.     

Bat colony size reduction coincides with clear-fell harvest operations and high rates of roost loss in plantation forest

Clear-fell harvest of forest concerns many wildlife biologists because of loss of vital resources such as roosts or nests, and effects on population viability. However, actual impact has not been quantified. Using New Zealand long-tailed bats (Chalinolobus tuberculatus) as a model species we investigated impacts of clear-fell logging on bats in plantation forest. C. tuberculatus roost within the oldest stands in plantation forest so it was likely roost availability would decrease as harvest operations occurred. We predicted that post-harvest: (1) roosting range sizes would be smaller, (2) fewer roosts would be used, and (3) colony size would be smaller. We captured and radiotracked C. tuberculatus to day-roosts in Kinleith Forest, an exotic plantation forest, over three southern hemisphere summers (Season 1 October 2006–March 2007; Season 2 November 2007–March 2008; and Season 3 November 2008–March 2009). Individual roosting ranges (100% MCPs) post harvest were smaller than those in areas that had not been harvested, and declined in area during the 3 years. Following harvest, bats used fewer roosts than those in areas that had not been harvested. Over 3 years 20.7% of known roosts were lost: 14.5% due to forestry operations and 6.2% due to natural tree fall. Median colony size was 4.0 bats (IQR = 2.0–8.0) and declined during the study, probably because of locally high levels of roost loss. Post harvest colonies were smaller than colonies in areas that had not been harvested. Together, these results suggest the impact of clear-fell harvest on long-tailed bat populations is negative.     

Effect of alpha-lipoic acid on oxidative stress,viability and motility in the common carp (Cyprinus carpio) spermatozoa after short-term storage and cryopreservation

As known for different metabolic functions, α-lipoic acid (ALA) has been tested for spermatozoa preservation of animals as well as of human, but not for fish spermatozoa. The present study determined the effects of ALA on short and long-term (cryopreservation) preservation of common carp (Cyprinus carpio) spermatozoa, for the first time. For that, spermatozoa were diluted in extenders containing 0 (control), 0.025, 0.05, 0.1, 0.5, 1, 2, 5, and 10 mM of ALA concentrations in both short-term preservation and cryopreservation. Spermatozoa motility parameters by computer-assisted semen analysis, viability, lipid peroxidation and catalase activity in spermatozoa were conducted in both 2nd and 120th hours of short-term storage and post-thaw samples. Higher percentages of total spermatozoa motility (80 ± 3) and viability (87 ± 3) were observed in 0.5 mM ALA group after 120 h of incubation. In post-thaw samples, higher percentages of these parameters were in 1 mM ALA group (74 ± 3 and 83 ± 2, respectively). Moreover, the results have shown that the addition of ALA until concentrations of 2 mM improved especially spermatozoa curvilinear velocity, maintained viability, and suppressed excessive lipid peroxidation during the preservations. In conclusion, the additions of 0.5 mM ALA for short-term preservation and 1 mM ALA for cryopreservation were the optimal concentrations, and shown the protective effects on common carp spermatozoa, when considering all measured parameters together.     

Cryopreservation of small leaf squares-bearing adventitious buds of <Emphasis Type="Italic">Lilium</Emphasis> Oriental hybrid ‘Siberia’ by vitrification

We report a new cryopreservation method for Lilium Oriental hybrid ‘Siberia’. Adventitious buds were induced from leaf segments cultured for 12 days on adventitious bud induction medium composed of half-strength Murashige and Skoog medium (MS) supplemented with 1 mg L^?1 α-naphthalene acetic acid and 0.5 mg L^?1 thidiazuron. Small leaf squares (SLSs, 3?×?4 mm), each bearing at least one adventitious bud, were cut from leaf segments, precultured on medium with 0.5 M sucrose for 1 day, and then treated for 20 min with a loading solution containing 0.4 M sucrose and 2 M glycerol, followed by exposure to plant vitrification solution 2 for 7 h at 0 °C. Dehydrated SLSs were directly immersed in liquid nitrogen for 1 h. Cryopreserved SLSs were re-warmed in MS medium containing 1.2 M sucrose for 20 min at room temperature, followed by post-thaw culture for recovery. With this procedure, 85% survival and 72% shoot regrowth were achieved following cryopreservation. The use of SLSs bearing adventitious buds for cryopreservation reported in the present study eliminates the time-consuming and labour-intensive step of shoot tip excision, and has great potential to facilitate cryopreservation in other plant species.     

Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation

Clove bud (Syzygium aromaticum) extract was added at concentrations of 0, 35, 75, and 115 μg/ml to ovine semen extenders in order to investigate the antioxidant activities of clove bud extract and its effects on semen quality parameters after cryopreservation of ram spermatozoa. The basic extender was composed of Tris, egg yolk, and glycerol. Two other extenders were prepared by substitution of egg yolk with either LDL or egg yolk + SDS. The DPPH inhibition test was employed to assess the antioxidant activity of clove bud extract. Results showed that, compared to vitamin E, clove bud extract had a higher antioxidant activity. Better sperm motility and movement characteristics (P < 0.05) were observed in the semen diluted with medium containing egg yolk + SDS than in that containing egg yolk and LDL. Progressive motility and movement characteristics of the sperm were significantly improved (P < 0.05) by adding 35 and/or 75 μg/ml of clove bud extract to semen extenders. Sperm viability and plasma membrane integrity were also higher (P < 0.05) in the semen exposed to medium containing egg yolk + SDS and 75 μg of clove buds extract after cryopreservation processes. Higher levels of clove bud extract, however, had adverse effects on all the sperm quality parameters and significantly reduced (P < 0.05) the motility, movement parameters, viability, and plasma membrane integrity of ovine sperm. It was concluded that the clove bud extract had an antioxidant potential that makes it useful for addition to semen extenders and that the best results are obtained with a maximum clove bud extract of 75 μg/ml. Moreover, the combination of egg yolk and a detergent was found to improve sperm quality after the cooling and freeze–thawing processes.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

UNUSUAL SHOOT GROWTH PATTERN IN A TROPICAL TREE,OREOPANAX (ARALIACEAE)

The tropical tree Oreopanax shows a number of characters usually considered as adaptations to a temperate climate combined with typically tropical features. This evergreen tree grows in flushes and forms “resting buds” between successive flushes. Because of the lack of marked seasons in the tree's habitat, the growth cycle shows almost no correlation with external factors. The terminal resting bud appears only after the rest period; new leaves within the bud are formed only during bud growth and bud break which last 10-14 weeks vs. 2-3 weeks for taxa in the temperate zone. Abscisic acid seems not to be involved in the induction of the rest period.     

Dormancy-associated gene expression in pea axillary buds.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs, PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots root apices; fully-elongated stems >elongating stems), and thus is an excellent “dormancy” marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs. Received: 10 December 1997 / Accepted: 23 January 1998