Multiple Shoot Development Research Articles

Development of efficient and scalable regeneration tissue culture method for Cannabis sativa

Large scale production of uniform disease-free plants is crucial for Cannabis sativa biotechnology. Existing micropropagation protocols rely heavily on shoot multiplication from existing meristems via direct organogenesis. Such protocols do not allow multiplication of plant material through continuous sub-culturing. Protocols that use indirect regeneration are usually not efficient enough and have very low multiplication rates. In the present study, an efficient protocol that uses a combination of direct organogenesis and callogenesis to induce multiple shoot development cultures is developed. Callogenesis was induced from various explants cultured on the media having various combinations of thidiazuron (TDZ) and naphthaleneacetic acid (NAA); best callogenesis and shoot regeneration was achieved from hypocotyl explants cultured on TDZ 0.4mgl-1 NAA 0.2mgl-1. Hypocotyls with cotyledonary node and shoot apical meristem were significantly better for shoot regeneration than explants without it. Shoots obtained from multiple shoot cultures were successfully rooted and then acclimatized under greenhouse conditions to develop into adult cannabis plants.

Strigolactone signaling inhibition increases adventitious shoot formation on internodal segments of ipecac.

SL inhibited adventitious shoot formation of ipecac, whereas the SL-related inhibitors promoted adventitious shoot formation. SL-related inhibitors might be useful as new plant growth regulators for plant propagation. In most plant species, phytohormones are required to induce adventitious shoots for propagating economically important crops and regenerating transgenic plants. In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), however, adventitious shoots can be formed without phytohormone treatment. Here we evaluated the effects of GR24 (a synthetic strigolactone, SL), SL biosynthetic inhibitors, and an SL antagonist on adventitious shoot formation during tissue culture of ipecac. We found that exogenously applied GR24 suppressed indole-3-acetic acid transport in internodal segments and decreased the number of adventitious shoots formed; in addition, the distribution of adventitious shoots changed from the apical to middle region of the internodal segments. In contrast, the SL-related inhibitors promoted adventitious shoot formation on both apical and middle regions of the segments. In particular, SL antagonist treatment increased endogenous cytokinin levels and induced multiple shoot development. These results indicate that SL inhibits adventitious shoot formation in ipecac. In ipecac, one of the shoots in each internodal segment becomes dominant and auxin derived from that shoot suppresses the other shoot growth. Here, this dominance was overcome by application of SL-related inhibitors. Therefore, SL-related inhibitors might be useful as new plant growth regulators to improve the efficiency of plant propagation in vitro.

Production of Salt Tolerant Thevetia peruviana Schum. Plants by Tissue Culture

Salt stress is a serious problem plant production and one of the main causes of damage on growth crops in the world. The plants are different in their ability to grow under salt stress. There are many studies to determine the plants salt tolerantce. This study was carried out in the tissue culture laboratory, Faculty of Agric., Saba Basha, Alex. Univ. to find a reliable protocol for in vitro propagation of Thevetia peruviana Schum. during the period from 2019 to 2020 was developed. Moreover, nodal explants were used during in vitro culture study for indication of multiple shoots and were inculated on various media with different combinations of NAA and KIN to study the effect on proliferation and development of multiple shoots, and the elongation of the newly formed on medium. The best medium for multiplication was a medium supplemented with3.00 and 4.00mg/l KIN and 0.50 mg/l NAA. Furthermore, the in vitro shoots showed healthy root development when the tested medium was supplemented with combination of 1.00 or 2.00mg/l IBA and0.50mg/l NAA. The newly formed shoot plantlets are selected for resistance to salt, and laboratory in vitro techniques proved very useful for this prupose. The plants showed good tolerance under low salt concentration at 2and 4gL-1 NaCl on multiplication of explants shootlets of thevetia plants with good survival. At the the salt concentration of 6gL-1, showed inhibitory effect on growth of plants with reduced the shootlets length, number of shoots, number of leaflets and number of roots/shootlets. Moreover, the proportion of carbohydrate content increased with the increasing salt concentration.

Effect of activated charcoal and phytohormones to improve in vitro regeneration in Vanda tessellata (Roxb.) Hook. ex G. Don

Vanda tessellata (Roxb.) Hook. ex G. Don. (Orchidaceae) is an epiphytic orchid species, commonly known as grey orchid, and explored enormously for its significant horticultural, medicinal, phytochemical and pharmacological properties. The present study describes an in vitro culture approach via asymbiotic seed germination assisted by phytohormones and enhanced proliferation of shoots and roots using activated charcoal for large scale production of V. tessellata. The maximum frequency of seed germination (100%) was achieved on Murashige and Skoog’s (MS) medium fortified with 1.5 mg L−1 indole-3 butyric acid (IBA). The seedlings developed from the protocorms were used for further proliferation and development of multiple shoots (24.8 ± 0.52 shoots per protocorm with 5.2 ± 0.35 cm average length) on MS medium augmented with 0.5 mg L−1 each of 6-benzylaminopurine (BAP) and indole-3 acetic acid (IAA) + 100 mg L−1 activated charcoal (AC) after 4th sub-culture. The optimal rooting of individual shoots was achieved on MS medium with 1.0 mg L−1 IBA. The well-developed plantlets were hardened using soilrite® + cocopeat + coconut husk complex in a greenhouse for 4 weeks. The plantlets were acclimatized under relatively increased temperature and low humidity for 4–5 weeks in the greenhouse with 98% survival rate. The protocol can be utilized for the development of strategies for large scale stable production of V. tessellata plantlets.

English

Considering the potential benefit of meristem culture in the mass production of quality propagation materials, an experiment was conducted to investigate meristem culture in KUWE (G1) and 223098 (G2) genotypes of anchote. Concentration levels of 6-benzylamimopurine (BAP) with and without gibberellic acid (GA3) and naphthalene acetic acid (NAA) for shoot induction while BAP and kinetin (Kn) for shoot multiplication were evaluated. Full and half nutrient-strength of Murashige and Skoog medium (FMS and HMS) with sole application of indole butyric acid (IBA) and indole acetic acid (IAA) were tested for root induction. About 80% (G2) and 71% (G1) of meristems induced shoots at 1 and 0.5 mg/L BAP, respectively. The highest shoot number (6.52), in G2, was recorded at 0.5 mg/L BAP while 0.5 mg/L BAP + 0.5 mg/L Kn produced 6.32 shoots in G1. FMS + 0.5 mg/L IBA was effective in 100% root induction in both genotypes. Survival rates of 58% (G1) and 42% (G2) were obtained. Generally, 0.5 mg/L BAP (G1) and 1 mg/L BAP (G) are best for shoot induction while 0.5 mg/L BAP (G2) and 0.5 mg/L BAP+0.5 mg/L Kn (G1) for multiple shoot development with best root formation on FMS+0.5 mg/L IBA. Key words: Direct regeneration, genotype, meristem explants, MS media, plant growth regulators (PGRs).

Impact of Kinetin and Benzyladenine on Growth Performance of Croton in Vitro

This study was carried out in the tissue culture laboratory, Faculty of Agric., Saba Basha, Alex. Univ. to fined reliable protocol for in vitro propagation of Croton (Codiaeum variegatun L.) during the period from 2019 to 2020 was developed. Moreover, nodal explants were used during in vitro culture study for indication of multiple shoots and inoculated on various media with different combinations of NAA as auxin and (BA & KIN) as cytokinin to study compare the effect of the two types of cytokinins on proliferation and development of multiple shoots, and the elongation of the new formed on medium. The best medium for multiplication was a medium supplemented with 2.00mg/l KIN and (0.50 or 1.00) mg/l NAA. Furthermore, the in vitro shoots showed healthy root development when the tested medium was supplemented with combination of 1.00mg/l KIN and NAA each, in turn. The new formed shoot plantlets (rooted plants) were acclimization ex vitro successfully. The survival rate of the ex vitro grown plants was 95%.

Optimization of regeneration using different plant growth regulator in Pomegranate (Punica granatum L.) cv.'Bhagwa' cultivar

A micropropagation technique is used for the pomegranate variety 'Bhagwa' with nodal segments as explants in present research. In current study, disease free culture were developed using a mixture of different concentrations sterilizing agents like carbendazim-50 per cent, cefotaxime, kanamycin, ketokenazol, and mercuric chloride (HgCl2). In addition to this, the phenolic exudates interfere with explant growth during In vitro condition. Consequently, the best approached proven to be transfer of explants at a regular interval of 24 h is best approach for phenolic exudates. Nodal segments were cultured on full strength Murashige and Skoog (MS) for shoot induction. The media was prepared supplemented with 6-benzylaminopurine (BAP), 0 to 2.5 mgl−1 and Kinetin (KIN), 0 to 1.0 mgl−1 produced maximum number of shoots (2.38±0.02) among all the treatments tested for shoot induction. Moreover, BAP with NAA proved to be best combination with adenine sulphate (30 mgl-1) and silver nitrate (1 mgl-1) for multiple shoot development gives produced more than 12 shoots (13.26±0.26) in 21 days intervals. Maximum number of root length and number of root recorded using ½ WPM with low concentration of IBA. The plantlets with well-formed root systems were progressively acclimatized in the greenhouse using soil, cocopeat (3:1) mixture and then transferred. Moreover, scanning electron microscopy (SEM) studies were carried out to observe and differentiate stomata and morphology of in vitro Pomegranate (Punica granatum L.) cv.'Bhagwa'

Application of lateral branching to overcome the recalcitrance of in vitro regeneration of Agrobacterium-infected pigeonpea (Cajanus cajan L.)

Establishment of a compatible and suitable regeneration and gene transformation protocol is of immense significance to achieve the desired improvement in pigeonpea through biotechnological approach. However, no reproducible protocol for large-scale generation of independent primary transformants plant lines through organogenesis virtually exists. Therefore, an attempt was initiated to develop a simple and straightforward protocol for easy generation of transformed lines through the facilitation of the production of multiple shooting and their lateral branching followed by selection of generated axillary buds. The protocol involves infection of longitudinally dissected cotyledonary node and shoot-tips of germinated seedling with Agrobacterium harboring genetic cassette having both the bialaphos-resistant and green fluorescent protein (gfp) genes as a plant selection marker and reporter gene respectively. The infected explants were allowed to grow in medium with 0.25 mg/L BAP and 0.1 mg/L gibberellic acid (GA3) for development of multiple shoots followed by micropropagation of nodal segments and selection under 4 mg/L bialaphos. The selected plants were found to contain integrated genes and carried through successive generations as revealed by dot blot, Southern hybridization, and expression of the reporter gene. In spite of having the scope of the generation of siblings, the resulted transformation frequency was found to be 6.66% irrespective of genotypes used in the present study as revealed by dot blot analysis. Overall, the protocol ensured generation of pigeonpea transgenic plants with considerable easy within a short time irrespective of genotypes/cultivars of the crop.

Role of Phytohormones and PGR's in Micropropagation of Anethum graveolens L. through Axillary Shoots and Evaluation of NaCl effect on Growth Parameters

An In vitro protocol is standardized for rapid in vitro propagation of Anethum graveolens through enhanced axillary shoot induction and optimization of role of BAP on shoot proliferation. MS basal medium augmented with various hormones (BA, NAA, KIN, and IBA) was used for multiple shoot development and rhizogenic response. The influence of various concentrations of NaCl was also observed on plant growth and morphological parameters. MS media fortified with BA (0.5–1.5) singly was induce axillary bud proliferation and in combination with NAA (0.1–0.25 mg L−1) and KIN (0.25–0.50 mg L−1), it was able to induce multiple shoots. BA at 1.0 mg L−1, KIN 0.5 mg L−1 and NAA 0.25 mg L−1 induced optimum number of multiple shoots (11.00 ± 0.379). The maximum frequency of rooting 83% with 3.67 ± 0.66 roots per explants was recorded on half strength MS medium supplemented with 1/2 MS + IBA 1.0 mg L−1 within 4 weeks of transfer of the shoots to the rooting medium. Hardening was successfully attained under partially controlled conditions inside the hardening room. The method could effectively used for conservation and clonal propagation to meet the pharmaceutical demands of this medicinally important plant species.

Effect of cytokinin combined elicitors (l-phenylalanine, salicylic acid and chitosan) on in vitro propagation, secondary metabolites and molecular characterization of medicinal herb – Coleus aromaticus Benth (L)

This study investigated the effect of cytokinin combined elicitors (l-phenylalanine, Salicylic acid and chitosan) on in vitro propagation of Coleus aromaticus. Furthermore, we determined the elicitor induced changes on genetic stability through Random amplified polymorphic DNA (RAPD), secondary metabolites and Phenylalanine ammonia lyase (PAL) gene expression. In in vitro propagation, the explant source of shoot tips and nodal explants were cultured on Murashige and Skoog (MS) medium with 0.5, 1.0, 1.5, 2.0 and 2.5mg/l of cytokinins (Benzyl amino purine (BAP), Kinetin (KIN)) for shoot bud induction. Further, nodal explants derived shoots cultured on the combination of 1.0mg/l of BAP with 0.5, 1.0, 1.5, 2.0, 2.5mg/l of l-Phe, 0.2, 0.4, 0.6, 0.8, 1.0mg/l of SA and 20, 40, 60, 80 of mg/l of Ch individually. The 79.81% of shoot bud induction was obtained with 1.0mg/l of BAP supplemented nodal explants. In multiple shoot development, 87% of multiple shoot development was obtained in the combination of 1.0mg/l BAP and 40mg/l chitosan supplemented culture. The developed shoots were rooted with 0.5mg/l of Naphthalene acetic acid (NAA) and transplanted to greenhouse condition with 75% survival rate. In vitro regenerated plants were subjected to RAPD profiling and it was exposed a similar banding pattern with that the mother plant. In secondary metabolites, compared to in vivo control the higher percentage of alkaloids (16), flavonoids (24), saponins (7.5), terpenoids (6.2), total phenolic content (24.4) and tannins (8) with increased PAL gene expression was analyzed in in vitro regenerated plants. Hence, this study shows that the choice of cytokinin combined elicitors supplementation during tissue culture noticeably influences not only multiple shoot development, but also the production of secondary metabolites without genetic modification.

In vitro Propagation and Ex vitro Acclimatization of Potato (Solanum tuberosum L.) Using Nodal Cutting Explants

Potato (Solanum tuberosum L.) is an economic tuberous crop cultivated worldwide in the temperate, tropical and subtropical zones. It occupies the fourth largest food crop following wheat, rice and maize. The aim of this study is to establish a protocol for in vitro initiation, multiplication, rooting and ex vitro acclimatization of potato plants (Solanum tuberosum L.) cultivars Lady Balfour and Bellini. This study was carried out in the plant tissue culture laboratory, the Faculty of Agriculture, Saba basha, Alexandria University, Egypt during the period from 2013 to 2016. An efficient and reliable protocol for in vitro propagation and ex vitro acclimatization of potato (Solanum tuberosum L.) was optimized. Nodal cutting explants were inoculated on various initiation or establishment media with different combinations of NAA and KIN and the neoformed shoots were cultured on proliferation (multiplication) media with different combinations of NAA and BAP for the development of multiple shoots, and the elongation media to elongate of the neoformed shoot. The subsequent elongated shoots were rooted, and acclimatized ex vitro, successfully. The best medium for shoot initiation was MS medium supplemented with 1.0 mg/l KIN. The favorable medium for multiplication was the tested medium augmented with 2.0 mg/l BA and 0.250 mg/l NAA. In addition, the most effective medium for elongation was the used medium enriched with 0.250 mg/l NAA. Furthermore, in vitro the shoots showed healthy root development when the tested medium was supplemented with combination of 1.0 mg/l IBA and 0.50 mg/l NAA (rooting stage).The combination of sand:perlite:peatmoss (1:3:3, v:v:v) was used as substrates for the hardening of the in vitro plantlets, as a potting mix, was the best suited mix for the acclimatization of plantlets ex vitro.

In Vitro Propagation and Ex Vitro Acclimatization of Magnolia (Magnolia grandiflora, Linn) Trees

This study was carried out in the tissue culture laboratory, Faculty of Agriculture, Saba basha, Alexandria University, Egypt during the period from 2013 to 2015. An efficient and reliable protocol for in vitro propagation of Magnolia grandiflora, Linnwas optimized. However, nodal explants from field grown of magnolia were used during in vitro culture study for induction of multiple shoots. Nodal explants were effectively surface sterilized with 30% Clorox (sodium hypochlorite) as commercial bleaches for 20 min plus 1.5mg/l mercuric chloride for 5 min with few drops of Tween-20, also. Nodal explants were inoculated on various initiation or establishment media with different combinations of IBA and KIN and the neoformed shoots were cultured on proliferation (multiplication) media for the development of multiple shoots, and the elongation media to elongate of the neoformed shoot. The subsequent elongated shoots were rooted, successfully. The best medium for shoot initiation was Woody Plant Medium (WPM) supplemented with 2.0 mg/l KIN and 1.00 IBA. The favourable medium for multiplication was the tested medium augmented with 5.0 mg/l KIN and 1.00 mg/l IBA.. Furthermore, the in vitro shoots showed healthy root development when the tested medium was supplemented with combination of 1.00 mg/l IBA and NAA ,each in turn (rooting stage). The shoots of Magnolia grandiflora multiplication and rooted successfully when they cultured in WP medium supplemented with 1.00mg/l charcoal. The combination of sand: compost (1:3) was used as substratum for the hardening of the in vitro plantlets, as a potting mix, was the best suited mix for the acclimatization of plantlets.

Development of an in vitro propagation method for the classified New York State-threatened native species Agrimonia rostellata

We developed an in vitro micropropagation protocol for Agrimonia rostellata Wallr. (Rosaceae), a New York State–threatened native plant species. For initial experiments, we investigated methods for disinfection of nodal sections from field-grown plants before transfer to culture medium to generate contaminant-free plant material. Fungal contamination was most prevalent. The only effective treatment was a combination of washes in a detergent solution followed by treatment with 70% ethanol, 10% bleach, plus vacuum infiltration with a solution of the antifungal compound Miconazole. We also found it was necessary to include 20 mg/l Miconazole in the culture medium to prevent fungal contamination. A Murashige and Skoog–based medium supplemented with 0.1 mg/l indole-3-butyric acid (IBA), 5 g/l charcoal, and 7 g/l agar was the only medium tested that resulted in both the development of multiple shoots and rooting. A starting population of 35 contaminant-free, in vitro plants was scaled up to 1013 plants in 6 mo. All rooted plantlets were successfully established in a soilless mix and then transferred to the field. These methods proved to be effective for generating a large population of A . rostellata for replicated field studies to identify the cause(s) of its decline.

In vitro plant regeneration in Capsicum chinense Jacq. (Naga Chili)

Article history: Received on: 07/01/2015 Revised on: 24/01/2015 Accepted on: 18/02/2015 Available online: 27/02/2015 An in vitro plantlet regeneration protocol was developed for Capsicum chinense Jacq. Naga Chili, one of the world’s hottest chili cultivars and an important horticultural crop of Northeast India. In vitro propagation plays a major role in conservation of genetically pure plants, crop improvement and production of disease free planting materials. The effect of different compositions of plant growth regulators on multiple shoot development and callus induction was investigated. Multiple shoot was induced by culturing explants in MS medium supplemented with Benzyl adenine (BA) in combination with Indole-3-acetic acid (IAA). Maximum numbers of shoot buds were induced in MS medium containing 5 mgl BA and 0.5 mgl IAA. Successful induction of callus from stem segments of in vitro raised plants were achieved in MS medium in combination with 3 mglBenzyl adenine (BA) and 1 mgl 1-Naphthaleneacetic acid (NAA).Shoot elongation and rooting were achieved in MS basal medium. This is the first successful report of plant regeneration from calluses in Capsicum chinense Jacq. Naga Chili. This protocol can be used as a cost effective method for the production of disease free planting materials and for genetic improvement and conservation of the crop.

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).

Development of high frequency multiple shoots in the yellow cactus, Selenicereus megalanthus

Development of high frequency multiple shoots in the yellow cactus, Selenicereus megalanthus

Factors affecting the efficiency of micropropagation from lateral buds and shoot tips of Rubus

Factors affecting micropropagation efficiency of 32 selections of Rubus, including the pre-treatment and initiation culture stages, were investigated. Chilling at 4°C for 6 weeks as a pre-treatment significantly promoted in vitro initiation culture; up to 65% of initiation cultures post chilling were successful. The cytokinin 6-benzyladenine (BA) was the most effective of the three tested for promotion of multiple shoot development in culture, with an average of three to seven shoots or plantlets developed from each single node. Alternating the concentration of BA between 4.44 and 13.31 μM from sub-culture to sub-culture improved recalcitrant Rubus micropropagation, and reduced the problems associated with long-term culture in the presence of high concentrations of BA. Reduction of the strength of macro- and micro-elements in the basal medium from half to one-third, with addition of 0.49 μM indole-3-butyric acid and 0.05% activated charcoal, and placing the cultures under reduced light intensity (17 μmol m−2 s−1), was found to alleviate chlorosis and improve micropropagation with high quality Rubus plantlets. Response to in vitro culture differed greatly and consequently different methods of micropropagation were required by different genotypes. From these selections, three types of micropropagation including micro-cutting, micro-shoots and multi-shoots were observed, and their efficiency was characterized.

Improving Multiple Shoot Proliferation in Bamboo Mosaic Virus-free Bambusa oldhamii Munro Propagation by Liquid Culture

An in vitro method for obtaining bamboo mosaic virus (BaMV)-free plantlets of Bambusa oldhamii Munro was developed. BaMV-free meristems were incubated on MS basal medium supplemented with 0.45 μm thidiazuron (TDZ) to induce the development of multiple shoots. Multiple shoot proliferation was higher in stationary liquid culture than on semisolid medium. Cytokinin was the key component for inducing proliferation, and TDZ was the stable and effective cytokinin for proliferation in long-term subcultures. Multiple shoots rooted after 1 month in MS basal medium containing 10.74 to 26.85 μm α-naphthaleneacetic acid with a rooting efficiency of 83%. Healthy, well-developed plantlets were transferred to soil in pots and raised in a greenhouse. Those plants derived from tissue culture were more vigorous than the ones derived from the traditional in vivo vegetative propagation method, air layering. The tissue culture-derived plants could produce the culms after 15 months. Fifteen of 38 plants flowered 2 years after being transplanted to the field.

(291) Plant Regeneration through Direct Somatic Embryogenesis in Homalomena `Emerald Gem'

Homalomena `Emerald Gem' is an important ornamental foliage plant and widely used for interior plantscaping. Current propagation of this cultivar has been primarily carried out through in vitro culture by organogenesis; regeneration through somatic embryogenesis has not been documented. This report describes successful plant regeneration via direct somatic embryogenesis from explants of different organs. Somatic embryos formed at and around the cut surface of petiole, spathe, and peduncle explants. Embryos also appeared at the base between expanded ovaries of the spadix segment, and around midrib of leaf explants. The optimal treatments for somatic embryo occurrence from petiole, spathe, and peduncle explants were MS medium containing 0.2 mg/L NAA or 0.5 mg/L 2, 4-D with 2.0 mg/L CPPU, and for spadix explants were MS medium with 0.5 mg/L PAA and 2.5 mg/L TDZ. Somatic embryos appeared 6 to 8 weeks after culture and formed large embryo clumps in 3 to 4 months. Somatic embryos produced more secondary embryos and geminated on induction medium. Multiple shoot development and plant regeneration occurred from somatic embryo clusters on MS medium without hormone or with 2 mg/L BA and 0.2 mg/L NAA. The regenerated plants grew vigorously after transplanting to a soilless container substrate in a shaded greenhouse.

Multiple leaders caused by the tarnished plant bug (Lygus rugulipennis) in Picea abies seedlings

Feeding by the tarnished plant bug (Lygus rugulipennis Popp.) has caused severe damage in Norwegian nurseries. Large quantities of seedlings have been removed after sorting. The life cycle of the insect has been well studied and documented. This study was a first attempt to investigate how the seedlings develop after the insect attacks. The first visible sign of damage is the development of multiple terminal buds. Two-year-old containerized seedlings were planted and followed for 4 years. Before planting, the seedlings were sorted into three damage classes: 0, undamaged; I, moderately damaged, up to 10 fully developed buds; and II, severely damaged, 10–20 small buds and no dominant apical bud. The damaged seedlings were somewhat shorter at planting but this difference disappeared with time. The seedlings in the two damage classes differed in their development of multiple shoots. At the end of the 4 year period about 50% of the seedlings in class II and about 20% from class I still had multiple leaders, or lateral stems or side branches that would probably produce spike knots in the future. The sorting routine currently used at the nursery was found to reduce the problem to an acceptable level for seedlings that were planted in the forest.