Elimination of five viruses from sugarcane using in vitro culture of axillary buds and apical meristems

Procedures were developed for the in vitro elimination of Sugarcane mosaic virus (SCMV), Sorghum mosaic virus (SrMV), Sugarcane streak mosaic virus (SCSMV), Sugarcane yellow leaf virus (SCYLV) and Fiji disease virus (FDV) from infected sugarcane. In vitro shoot regeneration, elongation and virus elimination through meristem tissue culture originating from both apical and axillary shoots were compared. The average rates of regeneration and elongation from apical meristem tissues were 91 and 66%, respectively, with the virus-free rate among elongated shoots ranging from 61–92%. Mature axillary buds were cultivated in vitro to produce axillary shoots, from which meristem tissues were excised and cultured. These meristem tissues regenerated (77–100%) and elongated (55–88%) in culture medium at approximately the same rate as the apical meristems. The average virus elimination rate was 90% among elongated shoots derived from mature axillary buds. All five viruses can be eliminated by meristem tissue culture from both apical and axillary shoots using a standardized procedure. The overall average efficiency of virus-free plant production was 45 and 58% from apical and axillary shoots, respectively. There were no significant differences for shoot induction or virus elimination when the meristems were harvested from either the apical or the axillary shoots. This is the first report of SrMV or SCSMV elimination from sugarcane, as well as elimination of any mixed virus infections. This new method of harvesting meristems from axillary buds greatly expands the amount of material available for therapeutic treatments and thereby increases the probability of eliminating viruses from infected sugarcane.     

Interrelations between respiration and dormancy in buds of three hardwood species with different chilling requirements for dormancy release

 Respiration in vegetative buds of mature Betula pendula, Alnus glutinosa and Prunus padus trees was measured monthly at 15°C from mid-October 1996 to natural outdoor budburst in April 1997. In B. pendula the effect of bud water content on respiration was also estimated (December–April) by artificial imbibition of buds for 24 h prior to measurement of respiration. For estimation of corresponding bud dormancy status, batches of twigs were forced at identical monthly intervals at 15°C in long days (24 h), and budburst recorded. In all species dormancy was deepest when the leaves were shed in October, and dormancy was first alleviated in P. padus followed by B. pendula and A. glutinosa. However, bud respiration capacity was not related to dormancy release as it decreased in all species from October to November and displayed no notable increase until February in P. padus, March in B. pendula and April in A. glutinosa, after completion of dormancy release. Rather, increase in respiration coincided with growth resumption prior to budburst. Artificial imbibition of B. pendula buds increased the water content by approximately 10% (FW) and induced a doubling of the respiration rate (December–February). Moreover, the seasonal variation in bud water content (October–April) explained 94% of the variation in respiration in B. pendula and P. padus, and 84% in A. glutinosa. These observations suggest an important role of water content for respiration. During a cold period from mid-December to mid-January with mean temperature of –9.7°C dormancy release was arrested in P. padus, and to some degree in A. glutinosa, whereas dormancy release progressed normally in B. pendula. This indicates species differences in lower critical temperatures for dormancy release. Received: 30 June 1997 / Acceped: 1 October 1997     

Acropetal disappearance of PsAD1 protein in pea axillary buds after the release of apical dominance

We recently isolated PsAD1 cDNA from pea (Pisum sativum L. cv. Alaska) seedlings, whose mRNA abundantly accumulated in dormant axillary buds and disappeared after decapitation [Madoka and Mori (2000) Plant Cell Physiol. 41: 274]. To further elucidate the function of PsAD1, we investigated the temporal and spatial distribution patterns of PsAD1 protein using Western blot and immunocytochemical analyses. Western blot analyses showed that accumulation patterns of PsAD1 protein in axillary buds after decapitation and in response to IAA and 6-benzyladenine were the same as those of PsAD1 mRNA. Immunocytochemical analyses showed that (1) PsAD1 proteins were localized in the procambia, leaf primordia, apical meristem, and secondary axillary buds in the dormant axillary bud, and this distribution was the same as that of PsAD1 mRNA, (2) PsAD1 proteins acropetally disappeared after decapitation, and (3) the growth of axillary buds occurred in the same manner. These acropetal changes occur in a manner similar to the way in which the procambium differentiates into vascular tissue. These results suggest that PsAD1 plays some role in the inhibition of growth and differentiation, or in the maintenance of the dormant state in axillary buds.     

Change in plasma membrane ATPase activity during dormancy release of vegetative peach-tree buds

Plant dormancy and dormancy breaking depend, at least partially, on peculiar short distance relationships between buds and tissues underlying buds (bud stands). In peach-tree, it was previously observed that dormancy was related to a high nutrient absorption capacity in tissues underlying buds. This situation could be linked to higher plasma membrane ATPase activity (EC 3.6.1.3), inducing a higher nutrient absorption, in bud stands. This work consists of characterization of the plasma membrane ATPase activity in vegetative buds and bud stands during the rest period and dormancy release. During the dormant period (October and November), plasma membrane ATPase activity was found to be higher in bud stands than in buds. This was correlated with a lower amount of plasma membrane ATPase in buds compared to bud stands during this period. Moreover, plasma membrane ATPase activation by trypsin treatment was not the same in both tissues and different levels of ATPase activation could be noted within the same tissue during the different stages of dormancy release. According to these results, it can be postulated that dormancy release in peach-tree, is related to modifications of plasma membrane ATPase properties in buds and bud stands during winter time.     

Encapsulation of micropropagated buds of six woody species

Regrowth after encapsulation in a sodium alginate matrix of micropropagated buds from six different in vitro proliferated woody species was evaluated. Actinidia deliciosa Liang & Ferguson (kiwifruit), Betula pendula Roth (birch), Crataegus oxyacantha L. (hawthorn), Malus spp. (apple), Rubus spp. (blackberry) and Rubus idaeus L. (raspberry) propagated in vitro were used as bud sources. Encapsulation with sodium alginate and subsequent regrowth on nutrient rich medium was compared to encapsulation with nutrient-enriched alginate capsules followed by regrowth on nutrientless medium. Apical and sub-apical buds of Malus (rootstock M. 27 and cultivar Starkspur Red) were also compared for encapsulation and regrowth ability. All species showed a regrowth after encapsulation, but only if cultured on enriched media. M.27 apical and sub-apical buds showed different regrowth ability after encapsulation with sodium alginate. Applicability of encapsulation of single micropropagated tree buds is discussed.     

Involvement of calcium signalling in dormancy release of grape buds

Artificial induction of grape bud dormancy release by hydrogen cyanamide (HC) serves as a reliable model system to explore the events occurring shortly after the induction of dormancy release. Recently, a group of genes with remarkable differences in expression level between HC-treated and control buds was identified. The identification of several calcium signalling-related genes within that group raised the hypothesis of the involvement of Ca(2+) signalling in grape bud dormancy release. Therefore, the effects of HC treatment on the expression profiles of several calcium sensors, the effect of the plasma membrane calcium channel blocker LaCl(3) and the calcium chelator EGTA on HC-induced and chilling-induced bud-break, and the effect of HC application on calcium-dependent protein phosphorylation activities in the bud tissue were studied. Here the HC-induced expression of Ca(2+)-ATPase is described, indicating that this treatment might evoke an increase in [Ca(2+)]cyt. Similar induction was confirmed for calmodulin, calmodulin-binding protein, and calcium-dependent protein kinase (CDPK). Both LaCl(3) and EGTA blocked the inducing effect of HC on bud-break, and their inhibitory effects were removed by supplying exogenous Ca(2+). Calcium-dependent histone phosphorylation was up to 70% higher in HC-treated buds. Endogenous protein phosphorylation assays detected four proteins exhibiting increased phosphorylation following HC treatment, of which two were phosphorylated in a calcium-dependent manner. One of these, a 47 kDa protein, presented strong and Ca(2+)-dependent phosphorylation only in HC-treated buds. The potential role of CDPK in the phosphorylation of this protein was supported by an immunoprecipitation assay. The data suggest, for the first time, that calcium signalling is involved in the mechanism of bud dormancy release.     

Abscisic acid catabolism enhances dormancy release of grapevine buds

The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)‐mediated repression of bud–meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up‐regulation of VvA8H‐CYP707A4, which encodes ABA 8′‐hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H‐CYP707A4 within grape buds, (b) the VvA8H‐CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H‐CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H‐CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H‐CYP707A4 level in the bud is up‐regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA‐mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.     

Structure and cytochemistry of the procambium in Salix buds during dormancy and dormancy breaking

This report presents a combined investigation of ultrastructural and enzymatic changes in the procambium from late winter to early spring. In January the procambial cells of dormant Salix buds have a convoluted plasma membrane with many plasmalemmasomes, numerous lipid bodies, large stacks of rough ER and plastids surrounded by smooth ER profiles. Several small lysosomes show activity of ATPase and acid phosphatases. In addition ER, nuclear envelopes, dictyosomes, and thylakoids have ATPase activity, and ER and plasmalemma, and nuclei also show acid phosphatase activity. In February metabolism seems to increase as indicated by lysosomes with membranous formations, dilated ER, nuclear envelopes, spiny vesicles, and polysomes. ATPase activity occurs in plasmalemma and vacuoles, and acid phosphatases in the middle lamella region of walls, in plasmalemma, vacuoles, ER, and nuclei. At the end of March, when growth starts inside the buds, but before they break, the stacks of rough ER disappear, and the vacuoles coalesce. Most of the lipid bodies have disappeared and the plastids have accumulated starch. Cell division and differentiation of procambial cells to protophloem and protoxylem have started. The distribution of ATPase increases; activity is found in walls and plasmalemma, and only a few small vacuoles still have ATPase and acid phosphatase activity. Notable is the appearance of ATPase in mitochondrial cristae and nucleoli and the occurrence of rather high levels also in endomembranes and dictyosomes.     

太白红杉顶芽与分枝格局的适应性分析

野外调查发现太白红杉 (L arix chinensis)枝条顶芽死亡比例较高 ,顶芽死亡对分枝格局产生较大影响 ,可形成 3种分枝类型 : 型、 型和 型。对 3种分枝类型枝条的芽数量、计盒维数以及植冠不同部位的分枝类型比例、顶芽死亡比例、主侧枝平均枝长和主侧枝总数量分别进行了统计分析。结果显示 ,芽数量 : 型 (115 .3) < 型 (15 4 .8) < 型 (2 0 9.9) ;计盒维数 : 型(1.30 5 ) < 型 (1.4 0 0 ) < 型 (1.5 37) ;顶芽死亡比例由树冠上层至下层逐渐提高 ,而冠层东南西北 4个方向的顶芽死亡比例无显著差异 ;主侧枝平均枝长由树冠上层至下层逐渐增加 ,而主侧枝总数量则逐渐降低 ;由于风、光照、坡度和坡向的影响 ,冠层4个方向间的主侧枝平均枝长和总数量均存在显著差异 ; 型分枝使植冠半径扩大 , 型分枝快速扩展植冠的横向空间 , 型分枝在扩展空间的基础上并实现对空间的有效占据。研究表明太白红杉枝条中一定比例的顶芽死亡增加了分枝形态的多样性 ,表现出顶芽和分枝格局的环境适应性 ,有利于提高树冠的空间占据能力     

The shoot apical meristem restores its symplasmic organization during chilling-induced release from dormancy

The shoot apex of overwintering perennials ceases its morphogenetic activity at the end of the growing season and transforms into a bud which is dormant and freezing-tolerant. In birch (Betula pubescens) these events are triggered by short photoperiod, and involve the production of 1,3-beta-D-glucan containing sphincters on the plasmodesmata. As a result, all symplasmic pathways shut down. Here we show that breakage of bud dormancy by chilling involves restoration of the symplasmic organization of the meristem. This restoration is likely to be mediated by 1,3-beta-D-glucanase, which was present in small spherosome-like vacuoles that arose de novo during dormancy induction. During chilling these vacuoles were displaced from the bulk cytoplasm to the cortical cytoplasm where they became aligned with the plasma membrane, often associated with plasmodesmata. At this stage the enzyme also appeared outside the vacuoles. During chilling, 1,3-beta-D-glucan disappeared from the plasmodesmal channels and wall sleeves, and the plasmodesmata regained the capacity for cell-cell transport, as demonstrated by microinjection of Lucifer Yellow CH and Fluorescein-tagged gibberellic acid. Collectively, the present experiments demonstrate that restoration of the symplasmic organization of the meristem is indispensable for the release of buds from dormancy and the assumption of a proliferation-competent state, and implicate 1,3-beta-D-glucanase action at the plasmodesmata. Based on these findings we propose a model for 'dormancy cycling' which depicts the meristem as passing through three sequential states of cellular communication with characteristic sensitivities to distinct environmental cues.     

剥鳞和激素处理对大樱桃花芽休眠解除及内源激素变化的影响

采用高效液相色谱法(HPLC)分析了剥鳞与激素处理对大樱桃花芽休眠解除及内源生长素(IAA)、赤霉素(GAD、玉米素(ZT)和脱落酸(ABA)变化的影响。结果表明,花芽中的ABA主要分布于鳞片内,鳞片中的GA3和ZT含量远低于去鳞芽,也低于完整芽。剥鳞能明显增加休眠花芽中内源GA2和ZT的含量,降低ABA的含量,对IAA含量的影响不大。剥鳞降低了ABA／GA3、ABA／ZT的比值,使花芽向促进生长、抑制休眠的方向转化。同时,休眠前、后期剥鳞均能明显提高萌芽率,中期剥鳞效果不明显。剥鳞后施用外源激素随休眠时期不同而有不同的破眠效果,早期剥鳞GA3的效果最好,6-BA次之,IAA最差;中期破眠效果不如早期,GA。和6-BA没有明显差别;后期以6-BA效果最好,其次是GA3和IAA;3次处理中ABA均明显抑制花芽萌发。     

Co-ordinated gene expression during phases of dormancy release in raspberry (Rubus idaeus L.) buds

Bud break in raspberry (Rubus idaeus L.) is often poor and uneven, with many of the subapical buds remaining in a dormant state. In order to determine the dormancy status of raspberry buds, an empirical measure of bud burst in a growth-permissive environment following exposure to chilling (4 degrees C cold storage) was developed. For cv. Glen Ample, percentage bud burst in intact canes and isolated nodes was recorded after 14 d. Isolated nodes (a measure of endodormancy) achieved 100% bud burst after approximately 1500 h chilling whereas buds on intact plants (combined endo- and paradormancy) required an additional 1000 h chilling. A microarray approach was used to follow changes in gene expression that occurred during dormancy transition. The probes for the microarrays were obtained from endodormant and paradormant raspberry bud cDNA libraries. The expression profiles of 5300 clones from these libraries were subjected to principal component analysis to determine the most significant expression patterns. Sequence analysis of these clones, in many cases, enabled their functional categorization and the development of hypotheses concerning the mechanisms of bud dormancy release. Thus a set of novel candidates for key dormancy-related genes from raspberry buds have been identified. Bud dormancy is fundamental to the study of plant developmental processes and, in addition, its regulation is of significant economic importance to fruit and horticultural industries.     

Physiological dormancy in seeds of tropical montane woody species in Hawai`i

Worldwide, there is relatively little information on seed dormancy and germination of tropical montane species. Our aim was to help fill this knowledge gap by conducting seed dormancy/germination studies on woody species from this vegetation zone in Hawai`i. All species had water-permeable seeds with a fully developed embryo. Seeds of 29 species (23 genera) were incubated in light/dark at 15/6, 20/10 and 25/15°C and germination monitored at 2-week intervals for 16–128 weeks. Seeds of Chenopodium oahuense, Dubautia menziesii and Silene lanceolata were non-dormant (ND) and those of 26 other species had physiological dormancy (PD); 10 of the 26 species had conditional PD. The optimum germination temperature regime(s) was (were) 25/15°C, 17 species; 25/10 and 20/10°C, 2; 20/10°C, 6; 20/10 and 15/6°C, 2; and 15/6°C, 2. Worldwide, PD in the woody genera included in our study is more common than ND. In addition to its contribution to the world biogeography of seed dormancy/germination, this study will be useful to conservation biologists who need to germinate seeds of tropical montane species.     

Mitochondrial response in the apical and lateral flower buds of the Hanfu apple to cold stress during the dormancy stage

In order to study the different physiological bases of cold tolerance in the apical flower buds (AFB) and the lateral flower buds (LFB) of the Hanfu apple (Malus domestica Borkh), we used 4-year-old grafted Hanfu plants as material and evaluated the physiological characteristics of mitochondria in the flower buds, such as electron transport chains (cytochrome pathway and alternative pathway), H₂O₂ content, mitochondrial membrane permeability transition (mPT), and MDA content. AFBs and LFBs showed different changes in total respiratory rate (V_t) during low-temperature stress, except that both reached the lowest V_ts at ?30 °C. The AFB V_t increased to a peak at ?25 °C and decreased sharply to its minimal value at ?30 °C, and then remained relatively low. In contrast, the LFB V_t decreased to its minimal value at ?30 °C and increased sharply to a peak at ?35 °C and then decreased again. In both AFBs and LFBs, the cytochrome pathway was still the main electron transport chain throughout the whole process, and the contributions of the cytochrome pathway (ρV_cyt/V_t) and of the alternative pathway (ρV_alt/V_t) showed similar tendencies to those of V_t as temperature changed. Changes in the AFB mPT were different from those of AFB V_t. LFB mPT zigzagged from peaks at ?25 °C and 35 °C. The H₂O₂ content of the LFBs increased from ?10 °C to ?30 °C, then decreased slightly from ?30 °C to ?35 °C, and then increased again. H₂O₂ content in AFBs went up steadily throughout the whole process. During the early stage of low-temperature treatment, before temperatures reached ?35 °C, LFB MDA content remained relatively low and later increased. MDA content in AFBs began to increase from the beginning of treatment. It can be concluded that the higher cold tolerance of LFBs relative to AFBs could be closely related to their higher V_t and ρV_alt/V_t, which may aid adaptations to stress by supplying energy and metabolic substrates under low-temperature stress conditions.     

Changes in the water status and rheological characteristics of pea seedling axillary buds during their transitions growth-dormancy-growth in apical dominance

The technique of isopiestic thermocouple psychrometry was used for the analysis of bud transition from dormancy to growth and back in 8-18-day-old pea (Pisum sativum L.) seedlings. We monitored changes in the water (ψ_w) and osmotic (ψ_{s + m}) potentials and also turgor pressure (ψ_p) in dormant buds and threshold turgor (Y) in growing buds, the latter being one of the cell-wall rheological characteristics. Seedling decapitation resulted in a decrease of Y in the bud, which coincided with the start of its outgrowth. The replacement of terminal shoot with exogenous auxin (IAA or NAA) retarded bud outgrowth and maintained the high level of Y, which argues for the auxin control of this parameter. When growth of the first axillary bud was inhibited by the second one, positioned higher and remained on the plant, the beginning of Y increase preceded visible correlative growth suppression; this makes this rheological index an early marker of bud transition from growth to dormancy. The effects of the terminal shoot part and auxin application on the bud osmotic status differed substantially. In fact, bud transition to dormancy in the presence of the terminal shoot, the main or developing from the second axillary bud, was accompanied by the rise in ψ_{s + m}, whereas, in the case of the replacement of the second bud with exogenous auxin, the first bud growth suppression occurred with the decrease in ψ_{s + m}. The low value of the bud ψ_{s + m} is a factor for creating a considerable gradient of the water potential between the stem and bud supporting water transport to the bud, which was much more active than in plants with a terminal shoot. It seems likely that this is the reason for the absence of complete growth suppression observed by us and other researchers even after application of high auxin concentrations. Immediately after seedling decapitation, ψ_{s + m} in the buds reduced; however, this was not the result of trophic metabolite redistribution due to the loss of their active sink because ψ_{s + m} reduced also in experiments with complete isolation of the bud releasing from dormancy in the chamber at 100% humidity. Auxin application to the cut surface of decapitated seedlings did not affect the ψ_{s + m} decrease. Like decapitation, cotyledon removal resulted in the increase in the bud turgor pressure. However, in this case, water stress did not change the bud osmotic status. Thus, the induction of osmotica accumulation in the bud after the removal of the terminal shoot is evidently related to neither trophic, nor auxin, nor hydraulic signal. The data obtained allowed us to conclude that both components of the bud water potential—ψ_{s + m} and Y—play an important role in the control of bud growth at apical dominance. Auxin produced in the shoot apex is involved in the control of Y, whereas the nature of the signal controlling the ψ_{s + m} level is unclear.     

桃花芽自然休眠期间水孔蛋白基因的转录表达

以10年生大田栽培及3年生盆栽曙光油桃花芽为试材,利用荧光定量PCR测定了油桃休眠及休眠解除期间(2009年9月15日-2010年1月15日)曙光油桃水孔蛋白基因δTIP1、PIP1;1的表达量,以及低温胁迫下的转录表达.结果表明:在油桃休眠及休眠解除期间,曙光油桃PIP1;1的转录水平呈现持续增高趋势,且1月的高水平表达使水分通过液泡膜和细胞质膜流出,减少了芽体水分含量,阻止细胞内冰晶的形成,从而抵御冻害;可溶性糖、可溶性蛋白、脯氨酸含量均达到最高,防止细胞的脱水伤害.低温处理2周后高水平表达说明PIP1;1为冷诱导基因.δTIP1的转录水平在休眠期间呈现波动性变化,至休眠解除时大幅度增高,这可能与休眠解除时,其上调表达被休眠解除信号及植物活性的增强所诱导有关.低温处理2周后,其表达没有升高,说明δTIP1并非冷诱导基因.