Axillary Shoot Development Research Articles

Meta-Topolin as an Effective Benzyladenine Derivative to Improve the Multiplication Rate and Quality of In Vitro Axillary Shoots of Húsvéti Rozmaring Apple Scion.

In vitro mass propagation of apple plants plays an important role in the rapid multiplication of genetically uniform, disease-free scions and rootstocks with desired traits. Successful micropropagation of apple using axillary shoot cultures is influenced by several factors, the most critical of which is the cytokinin included in the culture medium. The impact of medium composition from single added cytokinins on shoot proliferation of apple scion Húsvéti rozmaring cultured on agar-agar gelled Murashige and Skoog medium fortified with indole butyric acid and gibberellic acid was investigated. The optimum concentration for efficient shoot multiplication differs according to the type of cytokinin. The highest significant multiplication rate (5.40 shoots/explant) was achieved using 2.0 μM thidiazuron while the longest shoots (1.80 cm) were observed on the medium containing benzyladenine at a concentration of 2.0 μM. However, application of either thidiazuron or benzyladenine as cytokinin source in the medium resulted in shoots of low quality, such as stunted and thickened shoots with small leaves. In the case of benzyladenine riboside, the 8 μM concentration was the most effective in increasing the multiplication rate (4.76 shoots/explant) but caused thickened stem development with tiny leaves. In the present study, meta-topolin was shown to be the most effective cytokinin that could be applied to induce sufficient multiplication (3.28 shoots/explant) and high-quality shoots along with shoot lengths of 1.46 cm when it was applied at concentrations of 4 μM. However, kinetin was the least active cytokinin; it practically did not induce the development of new shoots. The superior cytokinin for in vitro axillary shoot development of apple scion Húsvéti rozmaring with high-quality shoots was the meta-topolin, but it may be different depending on the variety/genotype under study.

Tissue-specific expression of barnase in tobacco delays axillary shoot development after topping.

Shoot branching is an important agronomic trait that determines plant architecture and affects crop productivity (Shen et al., 2019). Molecular signals from the shoot apical meristem (SAM) create a hormonal environment that integrates with the expression of axillary bud-specific repressors such as BRANCHED1 (BRC1) to inhibit axillary shoot formation (Wang et al., 2019 and references therein). The signal is eliminated by topping (SAM removal), enabling the formation of new shoots (suckers) from axillary buds (Figure 1a).

Cranberry response to sulfentrazone rate, timing, and application volume

AbstractThe recent registration of sulfentrazone, a selective, soil-applied, PRE herbicide labeled for control of various weeds in cranberry, expanded the number of modes of action that could be used in the crop. A 2018 preliminary study in Massachusetts showed that high rates of sulfentrazone applied at the cabbage head stage reduced the number of flowering uprights (vertical stems) without impacting the final yield. To clarify the use patterns needed to promote crop safety when using sulfentrazone, six studies were conducted in New Jersey and Massachusetts in 2019 and 2020. Studies compared sulfentrazone applications made at two timings (spring dormant, SD; or cabbage head [CH] stage), two rates (280 and 420 g ai ha−1), and three application volumes simulating either chemigation (3,740 L ha−1) or boom application (190 L ha−1 alone or followed by 0.25 cm water wash-off). Boom application studies in New Jersey in 2018 and 2019 did not show significant long-lasting injury (necrosis or stunting). However, a comprehensive observation of cranberry uprights 8 wk after treatment showed a high rate of terminal bud necrosis, a reduction in the number of reproductive structures, and the development of axillary shoots associated with a high rate of sulfentrazone applied at CH. A mitigation study conducted in 2019 and 2020 confirmed the safety of chemigated sulfentrazone at the high rate with no yield reduction, regardless of crop stage at application. Washing off the herbicide from the cranberry canopy immediately after boom application did prevent the necrosis of terminal bud and the related development of nonproductive secondary shoots. Considering the results of this study, application of sulfentrazone over the top of cranberry vine before scales of the terminal bud start loosening would be prudent practice at this time.

Root and axillary shoot development of hairy vetch stem cuttings and cessation of flower development under a short photoperiod

AbstractClonal propagation is a valuable tool for plant breeders to maintain heterozygous genotypes in crop species that are not amenable to self‐pollination or apomixis. An example is the drought‐tolerant forage and N‐fixing cover crop hairy vetch (Vicia villosa Roth), which primarily outcrosses and is developed into composite cultivars by plant breeders. The ability to obtain clones of desirable genotypes in breeding nurseries would enable the maintenance of heterozygous genotypes for genetic study or the ability to reconstitute a true synthetic cultivar. We developed an efficient method of clonal propagation from stem cuttings to maintain genotypes in a vegetative state for a minimum of 2 yr, even when obtained from flowering parental plants. The long‐term viability of such clones is unknown, however. We determined that hairy vetch, which flowers under long days, exhibits a form of partial flowering where vegetative meristems are maintained during conditions that induce flowering. Clones obtained from flowering parents would resume reproductive growth and eventually senesce if kept in inductive conditions. However, if clones were grown under a continuous 6‐h photoperiod, reproductive development would cease and vegetative growth would resume indefinitely.

Overexpression of Populus transcription factor PtrTALE12 increases axillary shoot development by regulating WUSCHEL expression

The TALE (Three Amino acid Loop Extension) transcription factor family has been shown to control meristem formation and organogenesis in plants. To understand the functional roles of the TALE family in woody perennials, each of the TALE members of Populus trichocarpa was overexpressed in Arabidopsis as a proxy. Among them, the overexpression of PtrTALE12 (i.e., 35S::PtrTALE12) resulted in a dramatic increase of axillary shoot development with early flowering. Interestingly, expression of WUSCHEL (WUS), a central regulator of both apical and axillary meristem formation, was significantly increased in the 35S::PtrTALE12 Arabidopsis plants. Conversely, WUS expression was downregulated in 35S::PtrTALE12-SRDX (short transcriptional repressor domain) plants. Further analysis found that PtrTALE12, expressed preferentially in meristem tissues, directly regulates WUS expression in transient activation assays using Arabidopsis leaf protoplast. Yeast two-hybrid assays showed that PtrTALE12 interacts with SHOOT MERISTEMLESS (STM); however, the interaction does not affect the WUS expression. In addition, expression of both CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) genes was suppressed accordingly for early flowering 35S::PtrTALE12 Arabidopsis. Indeed, transgenic poplars overexpressing PtrTALE12 as well as Arabidopsis plants overexpressing AtBLH11, a close homolog of PtrTALE12, phenocopied the 35S::PtrTALE12 Arabidopsis (i.e., increased axillary shoot development). Taken together, our results suggest that PtrTALE12 functions as a positive regulator of axillary shoot formation in both Arabidopsis and poplar.

Controlling the barriers of cloning mature Picea abies (L.) H. Karst. via tissue culture and co-cultivation with Agrobacterium rhizogenes

Providing an improved protocol for cloning of Picea abies (L.) H. Karst. mature trees via Agrobacterium rhizogenes. In reforestation programs, rejuvenation of superior genotypes is of great importance in helping juvenile rootstock production. In this regard, the use of plant tissue culture to produce axillary and/or adventitious shoots from explants of mature trees has been the objective of many researchers. There are a few reports about the successful in vitro rooting of the regenerated shoots from explants of mature trees. There is a very low rate of natural rooting in conifers, between 1 and 5%. Furthermore, they are still hard to root even when auxins are applied. In this study, in vitro propagation of Norway spruce explants and its rooting improvement by A. rhizogenes with the aim of receiving an efficient clonal propagation method was investigated. The effects of BA (0.5, 1, 1.5, and 2 μM) and TDZ (0.0025, 0.025, 0.25, and 2.5 μM) were examined on axillary shoot development from shoot-tip explants of the Norway spruce. The effects of three strains of Agrobacterium rhizogenes (K599, AR15834 and GMI9534) were also examined on enhancement of the root induction. According to the results, after the treatment of 1.5 μM BA, 87.5% of shoot-tip explants produced axillary shoots (the highest percentage among treatments), and also the maximum mean number of regenerated shoots per explant (6.5) was achieved in this treatment. Furthermore, the induction of rooting was performed using IBA (7.5 µM) and IAA (0.5 or 1 μM) in the WPM medium along with keeping the explants in the darkness at 21 °C, which yielded 37.5% and 12.5% rooting, respectively. Moreover, the application of coumarin along with IAA caused an increase of 22% in rooting, compared to the use of IAA alone. Two strains, K599 and AR15834, succeeded in improving the percentage of rooting, with the highest being 54.2% as a result of using the K599 strain alongside 7.5 μM IBA. The proposed treatment can be used efficiently for the propagation of Norway spruce in landscaping and/or reforestation programs.

Sequence characteristics of Medicago truncatula cyclophilin family members and function analysis of MsCYP20-3B involved in axillary shoot development.

Cyclophilins (CYPs) belonging to the immunophilin family are present in all organisms and widely distributed in various cells associated with the activity of peptidyl-prolyl cis/trans isomerase. Plant CYPs are members of a multi-gene family and are involved in a series of biological processes. However, little is known about their structure, evolution, developmental expression and functional analysis in Medicago truncatula. In this study, a total of 33 CYP genes were identified and found to be unevenly distributed on eight chromosomes. Among them, 21 are single-domain and 12 are multi-domain proteins, and most were predicted to be localized in the cytosol, nucleus or chloroplast. Phylogenetic and gene structure analysis revealed seven segmental gene pairs, indicating that segmental duplication probably made a large contribution to the expansion of MtCYP gene family. Furthermore, gene expression analysis revealed that about 10 MtCYP genes (were) highly expressed involved in vegetative and reproduction tissues in M. truncatula, and MsCYP20-3B was mainly upregulated in stems, leaves and flower buds in alfalfa(Medicago sativa). Overexpression of MsCYP20-3B was shown to regulate axillary shoot development associated with higher jasmonic acid and abscisic acid contents in M. truncatula. Our study suggests the importance of the CYP genes family in development, reproduction and stress responses, and provides a reference for future studies and application of CYP genes for alfalfa genetic improvement.

Loss-of-Function of a Tomato Receptor-Like Kinase Impairs Male Fertility and Induces Parthenocarpic Fruit Set.

Parthenocarpy arises when an ovary develops into fruit without pollination/fertilization. The mechanisms involved in genetic parthenocarpy have attracted attention because of their potential application in plant breeding and also for their elucidation of the mechanisms involved in early fruit development. We have isolated and characterized a novel small parthenocarpic fruit and flower (spff) mutant in the tomato (Solanum lycopersicum) cultivar Micro-Tom. This plant showed both vegetative and reproductive phenotypes including dwarfism of floral organs, male sterility, delayed flowering, altered axillary shoot development, and parthenocarpic production of small fruits. Genome-wide single nucleotide polymorphism array analysis coupled with mapping-by-sequencing using next generation sequencing-based high-throughput approaches resulted in the identification of a candidate locus responsible for the spff mutant phenotype. Subsequent linkage analysis and RNA interference-based silencing indicated that these phenotypes were caused by a loss-of-function mutation of a single gene (Solyc04g077010), which encodes a receptor-like protein kinase that was expressed in vascular bundles in young buds. Cytological and transcriptomic analyses suggested that parthenocarpy in the spff mutant was associated with enlarged ovarian cells and with elevated expression of the gibberellin metabolism gene, GA20ox1. Taken together, our results suggest a role for Solyc04g077010 in male organ development and indicate that loss of this receptor-like protein kinase activity could result in parthenocarpy.

Analysis of nutrient deficiencies affecting in vitro growth and development of Eucalyptus dunnii Maiden.

Although basal medium optimization is a key factor in the success of tissue culture, its mineral composition is frequently disregarded when optimizing in vitro propagation protocols. A previous work on Eucalyptus dunnii micropropagation suggests that excessive callus formation and leaf chlorosis are related to specific nutritional conditions of the basal media. Recently, a novel basal medium based on the mineral nutrient analysis of E. dunnii young stump shoots was developed and successfully tested in plant regeneration and micropropagation of E. dunnii, avoiding all these issues. Considering this basal medium as an ideal growth condition, a mild deprivation of each macro and micronutrient and of the total organic fraction was imposed to E. dunnii in vitro cultures for 30d. As a result, K, Mg, Mn, Cl, Zn, Mo, Ni or Co deprivation quantitatively affected growth and development of axillary shoots. Moreover, leaf chlorosis and the development of organogenic callus under Fe deficiency, and leaf drop along with shoot tip necrosis under N deficiency were observed. These symptoms suggest that nutrient content in E. dunnii tissues needs to be above 420.3mgkg-1 for Fe and 27.7gkg-1 for N to avoidthe symptoms of leaf chlorosis and shoot tip necrosis. Additionally, the main role of Mn in quantitative responses and the antagonism between ions, especially for Mg/K and Mg/Zn, were denoted by the multivariate analysis. Overall, these results make a relevant contribution to the optimization of in vitro propagation of E. dunnii and other hard-to-propagate related species.

In vitro propagation of bamboo species through axillary shoot proliferation: a review

Bamboos, very relevant plants in many countries around the world, are propagated at large scale with extreme difficulties. Use of seeds is challenging because of plant’s sporadic flowering and long flowering cycles, together with seed recalcitrance and consumption by wild animals. Vegetative propagation of bamboo is mainly conducted by cuttings and by air layering. However, these methods are only useful at small-scale because they damage the mother plants, propagation material is bulky and difficult to be transported and is only available during few months of the year. Therefore, in vitro propagation offers the opportunity to obtain large progenies from elite genotypes. In most cases, when developing protocols for in vitro propagation of plants, specific conditions for individual species, genotypes and even development stages of the donor plants must be identified by trial-and-error experiments. Because of the size of and the large diversity observed in this plant family, it usually takes several months to define most adequate culture medium, combination of plant growth regulators and of other compounds for fostering the desired development in the explants. Therefore, in this detailed review, that also puts together results from hard-to-find literature, we list all identified cases, in which development of axillary shoots was used to propagate bamboo plants, by presenting successful ways for disinfection, in vitro bud sprouting, multiplication, rooting and acclimatization.

Ex situ potential conservation of ipecacuanha (Psychotria ipecacuanha (Brot.) Stokes.), a critically endangered medicinal plant species

P. ipecacuanha is a medicinal plant endangered in Colombia, due to the indiscriminate exploitation suffered several decades ago for its therapeutic importance. Its great medicinal value is due to emetine production and its derivatives, approached in the roots. Given these concerns, the aim of this research was to evaluate different parameters for in vitro conservation of P. ipecacuanha due to limited plant growth, using as in vitro explants plant nodes. In fact, plant growth response to 18, 23 and 25°C with and without addition to MS basal culture medium 15 and 30 gL-1 of mannitol osmotic regulator was evaluated. Registration of plant survival data (%), plant height (cm), plant development of axillary shoots, number of leaves, callus formation and vigour of developed buds and was initially performed at 3 months and then every two months until 12 months. The explants conserved for 9 months in culture medium without mannitol osmotic regulator and temperature of 18°C, showed response after transferring and reculturing in recovery culture medium for 6-8 weeks, this process had not achieved any effect on plant growth and vigour in the recovery phase. In addition, the critical parameters in the acclimatization process were determined.

Effects of endogenous hormones on variation of shoot branching in a variety of non-heading Chinese cabbage and related gene expression

Shoot branching (tillering) primarily determines plant shoot architecture and has been studied in many plants. Shoot branching is an important trait in non-heading Chinese cabbage (Brassica rapa ssp. chinensis Makino). The B. rapa ssp. chinensis var. multiceps exhibits unique and multiple shoot branching characteristics. Here, we analyzed the variation in shoot branching between ‘Maertou,’ with multiple shoot branching, and ‘Suzhouqing,’ a common variety. The levels of endogenous indole-3-acetic acid (IAA), zeatin riboside and active gibberellins in the shoot meristem tissues of the two cultivars were quantified by enzyme-linked immunosorbent assay during the vegetative growth stage. High levels of IAA maintained axillary bud dormancy and repressed axillary bud outgrowth allowing shoot branching to form in the vegetative stage in ‘Suzhouqing.’ In contrast, low levels of IAA did not inhibit axillary buds in ‘Maertou,’ while a high level of cytokinin promoted axillary bud growth and branch shoot development. Exogenous hormone (rac-GR24 and 6-benzylaminopurine) treatment showed that ‘Maertou’ was relatively sensitive to cytokinin, because the fold changes of cytokinin-responsive genes in ‘Maertou’ were significantly more frequent than those in ‘Suzhouqing’. Cytokinin was the direct regulator for axillary bud growth of ‘Maertou’. Compared with ‘Suzhouqing’, ‘Maertou’ was sensitive to cytokinin and this weakened the strigolactone–cytokinin branching pathway.

Analysis of bearing and branching behavior of two apricot cultivars

The nature of axillary production along shoots is one of the main factors influencing both vegetative and reproductive growth in apricot. This study aimed at analyzing the morphology and branching pattern of one-year-old shoots of 'Velkopavlovicka' cultivar in comparison with 'Bergeron'. 'Velkopavlovicka' is a classical, medium vigor, Czech cultivar which has naturally a spherical crown. In twelve-year-old trees grafted on apricot seedlings and pruned to an open vase, lateral branches were described from the second to the fourth orders. Each shoot was described as a sequence of metamers, and for each metamer the fate (latent, vegetative or floral) of the central bud as well as the number of associated flowers were noted. The presence or absence of development of axillary shoots from vegetative buds was also considered. A hidden semi-Markov chain was built for each cultivar on the basis of the sample of bivariate sequences corresponding to the two observed categorical variables, pooling all one-year-old annual shoots whatever the year. Both cultivars could be characterized by a similar organization in zones, but with different probabilities of occurrence of the median flower bud zone as well as by different mean number of central and associated flowers within this zone. Cultivar 'Bergeron' proved to be more floribund than 'Velkopavlovicka'. Moreover, they revealed biennial bearing in Central European conditions, but different reasons could be responsible for this behavior depending on the cultivar.

SlARF2a plays a negative role in mediating axillary shoot formation.

SlARF2a is expressed in most plant organs, including roots, leaves, flowers and fruits. A detailed expression study revealed that SlARF2a is mainly expressed in the leaf nodes and cross-sections of the nodes indicated that SlARF2a expression is restricted to vascular organs. Decapitation or the application of 6-benzylaminopurine (BAP) can initially promote axillary shoots, during which SlARF2a expression is significantly reduced. Down-regulation of SlARF2a expression results in an increased frequency of dicotyledons and significantly increased lateral organ development. Stem anatomy studies have revealed significantly altered cambia and phloem in tomato plants expressing down-regulated levels of ARF2a, which is associated with obvious alterations in auxin distribution. Further analysis has revealed that altered auxin transport may occur via altered pin expression. To identify the interactions of AUX/IAA and TPL with ARF2a, four axillary shoot development repressors that are down-regulated during axillary shoot development, IAA3, IAA9, SlTPL1 and SlTPL6, were tested for their direct interactions with ARF2a. Although none of these repressors are directly involved in ARF2a activity, similar expression patterns of IAA3, IAA9 and ARF2a implied they might work tightly in axillary shoot formation and other developmental processes.

Micropropagation of <i>Genista aetnensis</i> [(Raf. ex Biv.)DC

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

Micropropagation of Genista aetnensis [(Raf. ex Biv.)DC

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

DEVELOPMENT OF PROTOCOLS FOR MICROPROPAGATION OF ELITE GENOTYPE FORAGE ALLOGAMOUS LEGUME SPECIES

Forage legumes are essential components of pastures for livestock farms. Selecting the most high-yielding genotypes will increase biomass and seed production, increasing forage availability and pasture renewal. We developed a process to micropropagate high-yielding genotypes of the species Trifolium incarnatum, T. resupinatum, T. michelianum, T. subterraneum, T. isthmocarpum, T. vesiculosum, T. alexandrinum and sulla plants (Hedysarum coronarium). At the beginning of spring, stems from selected plants were collected and their axillary buds were cultured in vitro. Small stem segments with axillary buds were dissected and surface sterilized using a 20% (v/v) commercial bleach with detergent for 15-25 min, followed by washing with sterile distilled water. Axillary shoot development was induced by cultivating disinfected stem segments on MS basal salt and vitamins medium, supplemented with sucrose and benzyladenine and solidified with 0.7% (w/v) Microagar. The micropagated shoots were rooted on solidified MS medium without the addition of growth regulators. Most of the genotypes collected were successfully transferred to in vitro. Acclimatization to ex vitro conditions was not a limiting step. The produced plants will be further evaluated, in the greenhouse and in the field, to confirm the maintenance of the agronomical desirable traits.

In vitro propagation of Picconia azorica (Tutin) Knobl. (Oleaceae) an Azorean endangered endemic plant species

Picconia azorica (Tutin) Knobl. (Oleaceae) is a woody plant endemic from the Azores. It is considered as a part of a true relict taxon, reflecting its phylogeny, genetics and geography. Given the fragile status of this species, effective management and conservation are necessary for its survival and repopulation, initially for ecological reasons, but ultimately for forestry purposes as a valuable source of timber. Micropropagation is a useful tool for conservation programs, and in vitro culture techniques have, therefore, been developed for P. azorica by testing different combinations of media and growth regulators at each micropropagation stage. Adult plants from the field were successfully established in vitro on Rugini olive medium supplemented with 24.6 µM 2-isopentenyl adenine (2iP), resulting in 91.2 % sprouting of the nodal explants that survived the disinfection stage, although similar results were achieved using 18.2 µM zeatin. At the multiplication stage, the best shoot quality and the best compromise between shoot length and new axillary shoot development were achieved using the modified macronutrients Driver and Kuniyuki walnut medium supplemented with 24.6 µM 2iP and 0.6 µM indole-3 acetic acid. Rooting was most successful with half-strength macronutrients combined with 10 µM indole-3 butyric acid. Using this micropropagation protocol, a wide range of accessions were successfully established in vitro and are now being propagated on a larger scale for ecosystem restoration.

First report of mixed infection of Papaya ringspot virus and phytoplasma in papaya in India.

Papaya plants exhibiting symptoms such as multiple axillary shoots, leaf reduction, mosaic, chlorosis, flattened petiole, oily spots on stem and virescence of flowers were observed in Pune (India). Electron microscopy observations of leaf dip preparations revealed flexuous filamentous particle of 760x12 nm, suggesting the presence of a potyvirus, which was identified as Papaya ringspot virus (PRSV) in ELISA using a specific antiserum. The presence of PRSV was confirmed by RT-PCR using coat protein gene-specific primers that amplified a fragment of ca. 950 bp in size. Sequence analysis of the RT-PCR product (GenBank accession No. KJ142142) showed 87 to 100% identity at the nucleotide level with other Indian PRSV isolates (Jain et al., 2004). The presence of a phytoplasma was tested in symptomatic papaya by PCR using universal P1/P7 and R16mF2/R16mR1 primer pairs. BLAST analysis of the consensus sequence obtained by sequencing PCR products (GenBank accession No. JQ346525) revealed 99% nucleotide sequence identity with Candidatus Phytoplasma aurantifolia, a member of group 16Sr II phytoplasma. Transmission of the phytoplasma to healthy papaya grafted onto infected papaya was obtained four weeks after grafting with the development of multiple axillary shoots. The presence of a phytoplasma and a virus in papaya has previously been reported in Puerto Rico and Cuba (Licha, 2002; Arocha et al., 2009). To our knowledge, this is the first evidence of mixed infection of PRSV and a phytoplasma in papaya from India, although these two pathogens were reported separately earlier (Jain et al., 2004; Rao et al., 2011).

Micropropagation of cloudberry (Rubus chamaemorus L.) by initiation of axillary shoots

A method for micropropagation of the endangered <em>Rubus chamaemorus</em> L. (<em>Rosaceae</em>) from shoot tips was developed. Murashige and Skoog (MS) medium supplemented with BA and IBA in different concentrations was used for axillary shoot development. The most effective was MS medium containing 0.88 µM BA and 0.49 µM IBA with value of pH = 4.0. Cultures of microshoots (up to 12 shoots from a single explant) developed into regular shoots on the same medium or after transferring them onto MS media with 0.44 µM BA, 0.49 µM IBA and 434 µM SA. The rooting of initiated axillary shoots proved extremely difficult. A two-step procedure for root induction was adopted. Auxin- treated shoots were put to medium devoid of plant growth regulators. Rooted shoots were then transferred to pots and grown in the greenhouse until planted in the ground of the Botanical Garden.