Endogenous cytokinin distribution patterns at budburst in Granny Smith and Braeburn apple shoots in relation to bud growth

The possible relationship of branching habit to cytokinin content of apple shoots (Malus×domestica Borkh.) was investigated. One-year old apple shoots are acrotonic (distal branching), more strongly so in Granny Smith than in Braeburn. In the first trial, long, 1-year old Granny Smith and Braeburn apple shoots were sprayed on 29 August 1995 to break rest with dinitro-o-cresol (DNOC) oil (5%). The cytokinin contents of the xylem sap, the combined bark and buds, and the wood were determined in distal and proximal shoot halves over the next 6 weeks. Budburst (terminal and lateral buds) was first visible (green tip) in both cultivars on 20 September 1995. A greater increase in cytokinin content of distal xylem sap, coupled with elevated cytokinin in the distal wood, reflect the overall acrotony of both cultivars. The strong acrotony of Granny Smith is reflected in the higher cytokinin concentration in distal portion 1 week before the proximal portion of the shoot. The differential distribution of cytokinin reflects the pattern of budburst and may be correlated with growth habit. In a subsequent trial, Granny Smith shoots chilled and forced in the absence of roots showed an increase in cytokinin content of the bark and buds, and the wood as growth resumed. This was roughly comparable in magnitude to the increase observed under field conditions. The cytokinin increase in rootless shoots and differential distribution of cytokinin prior to sprouting, support the hypothesis that shoot-derived, rather than root-derived, cytokinins act to trigger spring budburst.     

Plum Rootstock Trials at East Malling

Summary

Dormant axillary buds excised from crowns of pineapple (Ananas comosus L., Merr.) cultured on growth regulator free Nitsch medium sprouted after 8–10 d. Sprouted buds produced multiple shoots (7–10 shoots per bud) upon transfer to solidified Murashige and Skoog medium supplemented with 9.67 μM NAA, 9.84 μM IBA and 9.29 μM KIN. Each isolated shoot upon subculture to liquid medium of the same composition further proliferated to form more multiple shoots (60–65 shoots) and were maintained on a gyratory shaker (90–100 rpm). In vitro grown shoots were rooted on White medium supplemented with 0.54 μM NAA and 1.97 μM IBA. In vitro plantlets were established in cups with soilrite and hardened for four weeks. Phenotypic variants such as albinos, white streaked shoots and shoots with elongated internodes were observed in in vitro cultures. Approximately 520 in vitro produced plantlets were established in the field and these plants exhibit somaclonal variation. Thirty-eight plants were found to be yellowish, spineless with anthocyanin streaks and three were anthocyanin rich, spined plants.     

Propagation of Bambusa vulgaris (yellow bamboo) through nodal bud culture

Summary

Bambusa vulgaris (yellow bamboo), is the most commonly cultivated and used bamboo species in many countries. With the increased demand for bamboo, the importance of bamboo plantations has been realized. This would require large quantities of planting material continuously for which tissue culture techniques offer a solution. The propagation of B. vulgaris through nodal-bud culture was studied. Single nodal segments were tested for bud-break and shoot growth on basic Murashige and Skoog (1962) medium (MS) supplemented with different combinations and concentrations of growth regulators. Results suggest that cytokinin is important for bud-break. Gibberellic acid enhances multiple shoot production in this species. The position of the node on the culm appears to affect bud-break and multiplication, middle nodes are the most suitable. Also, removal of prophylls enhances bud-break. The shoots developed from axillary buds could be rooted on MS basic medium at 50% macro and IBA (0.25 μ). Upon transfer to the field (after four weeks in the rooting medium), the shoots developed into true-to-type plants.     

Effects of high vineyard temperatures on the grapevine leafroll associated virus elimination from Vitis vinifera L. cv. Napoleon tissue cultures

In vitro culture of shoot tips and axillary buds was used for virus elimination from the Spanish autochthonous table grapevine cultivar Napoleon which was infected by Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine fanleaf virus (GFLV). High percentages of GLRaV-3-free plants (91–100%) were obtained by establishing shoot tip cultures from infected mother plants of the 29-228, 74-16 and 77-266 clones. Lower percentages of virus-free plants (71–87%) were obtained by in vitro culture of the first, the second and the third axillary buds of the growing distal shoots. Percentages of virus-free plants obtained with both shoot tips and axillary buds varied according to the time of the year when the explants were collected and the bud position on the shoot. A increased efficiency of in vitro tissue culture methods was observed when cultures were established in summer and it was due in part to the high vineyard temperatures reached in the southeast of Spain during the hot season. GFLV- and GLRaV-3-free plants were only obtained by thermotherapy in combination with tissue culture methods from co-infected plants of the clone 39-29.     

Transgene expression for Gladiolus plants grown outdoors and in the greenhouse

Transgene expression was evaluated for Gladiolus plants transformed with either the CaMV 35S, double CaMV 35S, rolD, or Arabidopsis UBQ3 promoter controlling the uidA or bean yellow mosaic virus coat protein gene in either the sense or antisense orientation to determine differences in expression for plants grown in the greenhouse and outdoors for two years. There was more variability in GUS expression when plants were grown outdoors than in the greenhouse for two years. Four of the six transformed plant lines with the UBQ3, rolD, and CaMV 35S promoters grown outdoors showed significant differences in GUS expression from year to year as compared to two of the six lines with the UBQ3 and rolD promoters grown in the greenhouse. When grown the same year, two plant lines with the CaMV 35S and one line with the rolD promoter showed 2–16× higher levels of GUS expression outdoors than in the greenhouse, and one plant line with the UBQ3 promoter had 31× higher GUS expression in the greenhouse instead of outdoors. Three of six plant lines transformed with the bean yellow mosaic virus coat protein gene in either the sense or antisense orientation under control of the double CaMV 35S promoter showed obvious transgene expression as compared to three lines that did not show expression or negligible expression for both years when plants were grown both outdoors and in the greenhouse. This study verified long-term gene expression, rather than silencing, for Gladiolus plants when grown outdoors and in the greenhouse from year to year.     

Chilling response of ‘Granny Smith’ apple lateral buds inhibited by distal shoot tissues

Apple (Malus×domestica Borkh.) shoots were decapitated before or after chilling and then forced to budburst to determine the influence of distal inhibition (paradormancy) on the chilling response of lateral buds. Shoots were chilled and forced with the presence or absence of an inhibitory distal disbudded shoot piece. Endogenous cytokinins were determined from distal and proximal segments of shoots segmented before and after chilling and/or forcing. The isolation of lateral buds from distal inhibition by decapitation before chilling resulted in a dramatic increase in growth rate. This increase is greater than that observed in terminal buds during the normal development of acrotony on intact shoots. Distal shoot tissues appear to inhibit the chilling response of lateral buds. On intact shoots cytokinin increased more in the distal shoot tissues, but in decapitated shoots cytokinin increased more in the proximal shoot tissues. Increased cytokinin in the proximal stem segment following decapitation is possibly associated with an increased lateral bud growth rate.     

Root induction in three species of bamboo with different rooting abilities

Rooting of in vitro axillary shoots from adult field culms of Bambusa atra, Dendrocalamus giganteus and D. hookeri and of juvenile seedling shoots of D. giganteus were investigated. B. atra rooted spontaneously without exogenous auxin during axillary shoot proliferation, while both Dendrocalamus species rooted only on transfer to rooting media with indole-3-butyric acid (IBA). D. giganteus required coumarin, an auxin protector, in addition to IBA. Rooting in adult shoots of D. giganteus was lower (45.6%) than that in the juvenile shoots (96.7%) but adult shoots of D. hookeri rooted well (88.9%). A pretreatment with thidiazuron (TDZ: N-phenyl-N-[(1,2,3-thidiazol-5-yl)urea]) induced development of axillary buds and subsequently 95% rooting in adult D. giganteus shoots but had no significant effect on rooting in juvenile shoots of D. giganteus or D. hookeri. Pretreatment with tri iodobenzoic acid (TIBA) arrested shoot and bud development leading to very low or no rooting in all three species.     

Micropropagation of guava (Psidium guajava L.)

Summary

Guava (Psidium guajava L.) is difficult to propagate using conventional asexual techniques, with most growers using seedling planting stock. However, these seedlings are highly variable. We therefore developed an in vitro technique to clonally propagate guava. Various concentrations of BAP (6-benzylaminopurine) and TDZ (thidiazuron) were used to regenerate and micropropagate plants. Two explant sources were compared: greenhouse grown plants (GHRP) and in vitro-harvested axillary buds (IVDS). GHRP with BAP at 2 mg l^–1 gave 3.7 shoots per single node cutting with an average length of 0.7 cm. Shoots 3.0 cm long were obtained with 0.5 mg l^–1 BAP, however only 2.1 shoots per explant were produced. For IVDS, the largest number of shoots (3.9 per explant) was obtained with BAP at 0.25 mg l–1 , with an average shoot length of 1.6 cm. Generally, lower concentrations of BAP gave fewer but longer shoots. The highest number of roots and longest roots per shoot (5.4 and 2.0 cm, respectively) were obtained with 1 mg l^–1 indole-3-butyric acid (IBA). A protocol for producing clonal plants over eight weeks is described.     

根癌农杆菌介导Cry1Ac基因转化‘雪青’梨获得转基因植株

 以‘雪青’梨叶片愈伤组织为外植体, 经根癌农杆菌介导将CaMV35S启动子调控下的Cry1Ac基因导入‘雪青’梨。698块愈伤组织和231个Kan^r芽与携带表达载体质粒pBX203的根癌农杆菌菌株LBA4404共培养3 d后, 转入含50 mg·L^-1 Kan的筛选培养基培养30 d, 15 d转接1次。结果表明,在MS + 5 mg·L ^-1 6-BA + 0.1 mg·L^-1 IAA筛选培养基中, Kan^r芽率为34.24% , 在1/2MS + 2 mg·L^-1 IBA+ 0.5 mg·L^-1 6-BA + 500 mg·L^-1AC筛选培养基中, 15.58% Kan^r芽生根。共获得再生植株32株, 经PCR和Southern-blot分析证明, 其中4株的基因组已成功导入和整合Cry1Ac基因。     

Molecular characterization of an S-adenosylmethionine decarboxylase gene from floral organ differentiating axillary bud in calamondin (× Citrofortunella mitis)

Summary

Calamondin (× Citrofortunella mitis J. Ingram & H. E. Moore) is known for its ability to develop floral organs to flower in the adult phase during the four seasons. To provide information on molecular events during floral differentiation from axillary buds, we constructed a cDNA library from floral differentiating axillary buds of adult phase calamondin. Ninety-six cDNA clones were randomly selected from this library and sequenced. Ninety-six nonredundant ESTs were identified. Two clones encoded S-adenosylmethione decarboxylase, a key enzymes in polyamine biosynthesis. CmSAMDC 1 (Citrofortunella mitis s-adenosylmethionine decarboxylase 1) contained an 1083 bp ORF that encoded a putative SAMDC precursor of 361 amino acids. Northern blot analysis revealed that CmSAMDC 1 was expressed in axillary buds prior to floral differentiation and in axillary buds immediately after floral differentiation, in immature flower (5 mm), in fully developed flowers (120 mm long), in floral parts (petals, pistils, and stamens) of trees in the adult phase of development, but not in leaves or axillary buds of juvenile phase nucellar seedlings or leaf tissue from trees in adult phase of development. In situ hybridization revealed expression of the CmSAMDC 1 gene in the floral apex of axillary buds after differentiation, and in the phloem of axillary buds and petioles. These results suggest that SAMDC could play a role in actively differentiating and developing reproductive and vegetative organs.     

The impact of within-row spacing on the productivity of glasshouse roses grown in two planting systems

Summary

Stem yield and quality of roses for cut flower production were evaluated. The plants were grown in two planting systems as an alternative to the traditional ``vase-shaped'' system. In the trellised system, two cultivars of Rosa hybrida (cvs Gabrielle and Kardinal) were planted in a commercial glasshouse in 3.m sections of bed. Within-row spacing was varied to give 6–16 plants m<sup>–2</sup>. After a three-month establishment phase the basal shoots were bent to an angle of 308 above horizontal and restrained with a trellis wire. Flowering shoots sprouting from axillary buds along a basal shoot were harvested at their lowest node, minimizing branching. Compared with ``vase-shaped'' rose plants at the same density, trellised roses produced 24% more basal shoots, 46% more flowering shoots (cv. Gabrielle) and approximately 46% less blind shoots per plant over five months. Phenotypic variation was greater in cv. Gabrielle than in cv. Kardinal in response to within-row spacing, as indicated by the number of basal shoots formed. Within-row spacing, over the range explored, did not affect the number of flowering shoots per basal shoot. Trellising rose plants increased stem yield and quality, but production over the long-term requires further investigation. The single shoot planting system contained a mixed population of single-stemmed rose plants of Rosa hybrida (cvs Gabrielle and Gerdo). It was grown in a field over a range of within-row spacings to give 20–105 plants m^–2. Over three harvests, increasing the number of plants by 10 plants m^–2 reduced the proportion of flowering shoots by 4.4%. Expressed on a unit area basis, a five-fold increase in plants m^–2 produced a three-fold increase in stem production.     

In vitro micrografting of apical and axillary buds of cacao

This study aimed to evaluate in vitro grafting of Theobroma cacao where seedlings of the UF 677 genotype were used as the rootstock and apices or axillary buds of a Trinitarian genotype were used as scion. Three methods of grafting using scions from seedlings were evaluated. Apical grafts using apex and side grafts using apex displayed better graft success (95 and 80%, respectively). However, side grafts using axillary buds reached a greater height on average and a higher number of leaves per plant (1.76 cm and 3.72, respectively). Histological studies revealed new vascular elements at the graft union area. Side grafts with axillary buds provided the highest survival rate (82%) after the acclimatization step. A shoot of at least 1 cm with two leaves is required for plant survival after transfer to ex vitro conditions. Side grafting was carried out with axillary buds from adult trees and nursery plants. Only the grafts with buds from nursery grafted plants were successful, with a rate of 26%. Overall, side grafting with axillary buds is the most appropriate method for cacao micrografting. This method can be used for clonal propagation and for the establishment of in vivo and/or in vitro cacao germplasm collection.     

The use of CPPU for efficient propagation of pineapple

The use of forchlorfenuron (N-(2-chloro-4-pyridyl)-N′-phenylurea) (CPPU) for efficient propagation of pineapples was investigated. About 85% of axillary buds can be forced to sprout by soaking defoliated stem pieces (12 cm in length) in a 2.5 or 5.0 mg l⁻¹ CPPU solution for more than 3 h. The use of old stems taken from the third or fourth ratoon plants had the advantage of less liability to fungal decay, as compared to young stems from the first crop plants. The CPPU treatment combined with the removal of shoots from stems at monthly intervals significantly increased the number of shoots per stem piece (about 15 shoots per piece at 5.0 mg l⁻¹ CPPU), and resulted in a more uniform shoot size (the percentage of shoots within a range of 5–15 cm in length was about 90% at 5.0 mg l⁻¹ CPPU). The rooting of shoots was easily promoted within 1 month by treating the basal portion of shoots with 20 mg l⁻¹ indole-3-butyric acid (IBA) for 15 min. The CPPU method required about 5 months for plantlet propagation. From these results, we found that pineapple plantlets could be efficiently propagated by the following method: (1) soaking defoliated stems in a 2.5–5.0 mg l⁻¹ CPPU solution for more than 3 h; (2) harvest of developed shoots from the stems at regular intervals; and (3) promotion of rooting on the shoots at 20 mg l⁻¹ IBA for 15 min.     

灵武长枣高效继代增殖体系的建立

对灵武长枣茎段离体培养、高效继代增殖体系进行了研究。结果表明:以灵武长枣当年萌发幼嫩枣头为外植体,诱导腋芽萌发,细胞分裂素/生长素的比值为5倍时,腋芽萌发率最高;将萌发的嫩梢进行继代增殖培养,继代增殖茎段不同的放置方式、茎段不同位置、培养基的相态对增殖倍数有影响。     

Effect of maleic hydrazide on endogenous cytokinin contents in lateral buds,and its possible role in flower bud formation on the Japanese pear shoot

In order to elucidate the role of maleic hydrazide (MH, 1,2-dihydro-3,6-pyridazinedione; coline salt) in increasing flower bud formation on the Japanese pear, the effect of MH on cytokinin contents in the lateral buds of Japanese pear shoots were investigated. Foliar application of MH at 2600 mg l⁻¹ increased zeatin, zeatinriboside, and isopentenyladenine levels in lateral buds though isopentenyladenosine concentration decreased. Application of 2,3,5-triiodobenzoic acid (TIBA), an inhibitor of polar auxin transport, also increased the endogenous cytokinin levels in lateral buds. It is supposed that these increases of cytokinin in lateral buds may be involved in the increase of flower bud production on the Japanese pear shoot. The increases of cytokinin induced by these chemicals may be caused via the depletion of the auxin level and/or the activity in shoot tissues.     

Castanea sativa plantlets proliferated from axillary buds cultivated in vitro

Chestnut plants were proliferated in vitro from axillary buds of juvenile shoots. N6-Benzyl-aminopurine (BAP) at 0.1?0.5 mg l^?1 was optimal for shoot multiplication. The important role played by the macronutrient formula on shoot multiplication, and especially on the rooting-stage, is emphasized. The MS ($\text{1}\text{2}$ NO₃) macronutrients gave the best rooting percentage as well as the highest number of roots per rooted shoot. In these experiments, shoots remained in the 3 mg l^?1 indole-3-butyric acid (IBA) medium for 12 days, after which they were transferred to an auxin-free medium where roots developed fully. Optimum rooting was achieved by immersing the 1 cm basal end of shoots in concentrated IBA solutions (0.5?1 mg ml^?1) for periods ranging from 2 to 15 min.     

Evaluation of an alternative D-amino acid/DAAO selection system for transformation in apple (Malus × domestica Borkh.)

Summary

The conventional selection system for apple transformation is based on the selectable marker gene, nptII, encoding antibiotic resistance against kanamycin. We tested an alternative selection system based on the use of D-amino acids using the gene, D-amino acid oxidase 1 (dao1) as the selectable marker, in order to avoid the presence of antibiotic resistance genes in the resulting transgenic apple plants. In addition, dao1 allowed the selection as well as the elimination of dao1-transgenic plants, based on differences in the toxicity of different D-amino acids. Regeneration experiments using apple leaf explants revealed that 2 mM D-serine or D-alanine inhibited shoot regeneration. We performed transformation experiments using the apple cultivars ‘Gala’, ‘Holsteiner Cox’, and a progeny of the apple cultivar ‘Pinova’, and the vector p35S::dao1-intron, containing the dao1 and nptII selectable marker genes. Several shoots regenerated successfully on selection media containing various concentrations of D-serine or D-alanine, but transgenic shoots were not obtained. However, three dao1/nptII transgenic apple lines were obtained after selection with kanamycin, indicating that the vector was functional. Furthermore, we showed that 20 mM D-serine could be used to select dao1-transgenic shoots from non-transgenic in vitro shoots, whereas 13 mM D-isoleucine had the opposite effect.     

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.     

In vitro micropropagation of the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck and effects of sprayed GA3 after transplantation to ex vitro conditions

We established the conditions to micropropagate the ornamental prickly pear cactus Opuntia lanigera Salm–Dyck through axillary shoot development from isolated areoles. For the shoot proliferation stage different explant orientation (vertical and horizontal), type of cytokinin (BA, DAP and K), and concentrations (0, 1.25, 2,5, 5.0 and 7.5 mg/L) were evaluated. Media [Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Phys. Plant. 15, 473–497: 50 and 100%], and carbohydrate concentration (0.25, 0.5, 0.75 and 1.0%) were studied to optimize individual shoot growth and elongation. Following micropropagation and plantlet acclimatization, the effects of GA₃ on plant growth were determined by spraying a series of increasing concentrations (0, 150, 300 and 450 ppm). A reliable and efficient protocol of micropropagation was established for this particular plant species. The greatest propagation ratio (shoot proliferation) was obtained when explants were cultured in vertical orientation (4.975 shoots per explant) as compared to horizontal position (3.692 shoots per explant). The addition of BA to the media resulted in increased shoot number per explant (8) in comparison to K and DA, which produced only 2 shoots in average. However, after 42 days of culture, significantly higher shoot length was obtained with DAP (14 mm) compared to K and BA (4 mm). After the shoot proliferation stage, an elongation subculture was performed prior rooting in which shoot growth was enhanced when crowns of shoots were cultured in 50% of basal salt formulation of Murashige and Skoog (1962) and low sucrose concentration (2.5 and 5%). Exogenous application of GA₃ after plantlet acclimatization on glasshouse conditions increased spine-hair (developed from areoles in young plants) length as part of short-term effects. However, significantly higher values were obtained in plantlets treated with 300 ppm of GA₃ when compared with the rest of the treatments. At the end of the study, the most important long-term effect produced by GA₃ was the suppression of total shoot growth. The micropropagation protocol described here and the conditions to grow the plants through fertigation plus the application of GA₃ that induced changes in the phenotype may be used in commercial exploitations to regenerate 12,500 plantelts in average after 12 months of culture and produce healthy plants with better ornamental characteristics and higher commercial value.