Effects of 6-benzylaminopurine on photosystem II functionality and leaf anatomy of <Emphasis Type="Italic">in vitro</Emphasis> cultivated <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis>

Cytokinins (CKs) are often used during the in vitro cultivation of plant species. However, it is not clear how CKs, such as 6-benzylaminopurine (BAP), affect photosystem PS) II functionality and leaf anatomy over a long period of in vitro plant propagation. The aim of this study was to analyze the residual effects of BAP on the photosynthetic performance and leaf anatomy of Aechmea blanchetiana after 120 d without exposure to CKs. Aechmea blanchetiana plants previously grown in vitro were transferred to Murashige and Skoog (MS) culture media containing 0, 5, 10, 15, or 20 μM BAP. After 60 d on the MS medium with BAP, explants were subcultivated twice on the MS medium without growth regulators, first in a stationary liquid medium for 60 d and then in a solidified medium with 6 g dm^-3 agar for 60 d. Leaf anatomy, pigment content, and chlorophyll a fluorescence were assessed for plants from each treatment after 120 d on the CK-free medium. Stomatal density presented a negative linear correlation with BAP concentration. Pigment content decreased in plants subjected to previous BAP exposure. An increase in absorbed energy flux per reaction center (ABS/RC) and a sharp decrease in energy transport flux (ETo/RC) followed by an increase in energy dissipation flux (DIo/RC) also occurred. Furthermore, maximum quantum yield (F_V/F_M) decreased as a function of BAP concentration. Thus, the use of BAP during in vitro propagation of A. blanchetiana induced long-term physiological defects.     

Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds

Cypripedium flavum, known as the rare lady’s slipper orchid, is one of the endemics with a yellow flower in China. Due to its conservation and commercial requirement, establishment of an efficient method for micropropogation is urgently needed. Multiple shoots were obtained by placing seedlings from seeds of C. flavum on Harvais media supplemented with two cytokinins (BAP or KIN) used alone or in addition to different concentration of potato homogenate. The effect of BAP was better than that of KIN on shoot multiplication. The Havais media supplemented with BAP (2.22 μM) and potato homogenate (20 g l⁻¹) was the most effective, providing high shoot multiplication frequencies (95%) associated with a high number of shoots per explant (2.55 shoots/plant). For root formation, high rooting and survival were achieved using 1/2 Harvais media supplemented with 0.6 g l⁻¹activated charcoals. High-level activated charcoal increased the number and the length of roots because the activated charcoal could absorb BAP in the media. This study demonstrated that C. flavum could be micropropagated by using multiple shoots of seedlings derived from mature seeds.     

Effect of 6-Benzylaminopurine on the Structure of the Photosynthetic Apparatus of Faba Bean (Vicia faba L.)

The level of endogenous cytokinins changed with growth and development of faba bean (Vicia faba L.) leaves. Typical of juvenile leaves, amounting to 25% of the final leaf size (S _max), was a low content of these plant hormones. The level of cytokinins increased in growing leaves (50% of S _max) and decreased in leaves that stopped growing. The content of cytokinins in senescent leaves dropped considerably. Exogenous treatment with 6-benzylaminopurine (BAP) had no effect on the structure of the terminal phloem; however, it did (1) stimulate elongation of mesophyll cells; (2) increase the area and thickness of the leaf blade, amount of photosynthetic pigments, and assimilation potential; and (3) delay senescence of the leaves and defoliation, thereby increasing the biomass of the aboveground plant part. It is inferred that BAP has the potential for inducing the development of photosynthetic apparatus and increasing the yield of the green mass of faba bean.     

Effect of cytokinins on <Emphasis Type="BoldItalic">in vitro</Emphasis> development of autotrophism and acclimatization of <Emphasis Type="Italic">Annona glabra</Emphasis> L.

The role of cytokinins in the differentiation of the photosynthetic apparatus in micropropagated plants and their effect on the plant’s ability to transition from a heterotrophic to an autotrophic condition during acclimatization was investigated. Annona glabra L. shoots were cultured on woody plant medium supplemented with sucrose and different cytokinins to evaluate leaf tissue for chloroplast development, chloroplast numbers, photosynthetic pigmentation, total photosynthetic potential, and soluble sugar content. Plants were transferred to the rooting medium in the presence or absence of sucrose and then acclimatized. Kinetin and benzyladenine (BAP) stimulated chloroplast differentiation. Inclusion of zeatin in the medium induced the formation of greater numbers of chloroplasts in the leaves, while plants cultivated in the presence of only kinetin and BAP demonstrated greater chlorophyll a and carotenoid content. The use of kinetin and BAP during in vitro culture promoted accumulation of dry matter during the acclimatization phase, especially in plants rooted under autotrophic conditions (without sucrose). Kinetin and BAP promoted development of more leaf area and greater plant survival rates in plant acclimatization on both autotrophic and heterotrophic media. The inhibitory effects of thidiazuron on the differentiation of chloroplasts, accumulation of chlorophyll a, and photosynthetic potential were examined.     

Cytokinin-induced changes in the chlorophyll content and fluorescence of in vitro apple leaves

Cytokinins (CKs) are one of the main regulators of in vitro growth and development and might affect the developmental state and function of the photosynthetic apparatus of in vitro shoots. Effects of different cytokinin regimes including different types of aromatic cytokinins, such as benzyl-adenine, benzyl-adenine riboside and 3-hydroxy-benzyladenine alone or in combination were studied on the capacity of the photosynthetic apparatus and the pigment content of in vitro apple leaves after 3 weeks of culture. We found that the type of cytokinins affected both chlorophyll a and b contents and its ratio. Chlorophyll content of in vitro apple leaves was the highest when benzyl-adenine was applied as a single source of cytokinin in the medium (1846–2176 μg/1 g fresh weight (FW) of the leaf). Increasing the concentration of benzyl-adenine riboside significantly decreased the chlorophyll content of the leaves (from 1923 to 1183 μg/1 g FW). The highest chl a/chl b ratio was detected after application of meta-topolin (TOP) at concentrations of 2.0 and 6.0 μM (2.706 and 2.804). Chlorophyll fluorescence was measured both in dark-adapted (F_v/F_m test) and in light-adapted leaf samples (Yield test; Y(II)). The maximum quantum yield and efficiency of leaves depended on the cytokinin source of the medium varied between 0.683 and 0.861 (F_v/F_m) indicating a well-developed and functional photosynthetic apparatus. Our results indicate that the type and concentration of aromatic cytokinins applied in the medium affect the chlorophyll content of the leaves in in vitro apple shoots. Performance of the photosynthetic apparatus measured by chlorophyll fluorescence in the leaves was also modified by the cytokinin supply. This is the first ever study on the relationship between the cytokinin supply and the functionability of photosystem II in plant tissue culture and our findings might help to increase plantlet survival after transfer to ex vitro conditions.     

Ontogenetic Changes in the Photosynthetic Apparatus and Effects of Cytokinins (Review)

Data on structural and functional changes in the photosynthetic apparatus of agricultural plants are presented. Results of experiments with the regulatory effects of cytokinin-like phytohormones on photosynthesis are discussed. Cytokinins are involved in the structural formation and maintenance of the photosynthetic apparatus, stomatal function, supply of CO₂ to carboxylation sites through the leaf mesophyll, synthesis of pigments and enzyme systems, and regulation of photoreduction and carbon metabolism. Possible mechanisms mediating the regulatory effects of cytokinins are discussed.     

In vitro propagation,clonal fidelity and phytochemical analysis of <Emphasis Type="Italic">Rhodiola imbricata</Emphasis> Edgew: a rare trans-Himalayan medicinal plant

The present study focuses on development of a micropropagation protocol for true to type plants of Rhodiola imbricata, an endangered medicinal plant found in trans-Himalayan Leh-Ladakh region of India. It also aims at analyzing the pharmaceutically important secondary metabolites and antioxidant potential of in vitro and in vivo plants. Various cytokinins and auxins were tested for shoot proliferation and in vitro rooting of the microshoots, respectively. Random primers were used for checking genetic uniformity at different stages of micropropagation. Pharmaceutically important secondary metabolites of R. imbricata such as Rosavin, total polyphenols and free radical scavenging activity were analyzed by HPLC. Among different cytokinins used, BAP (5 µM) and TDZ (1 µM) were found to perform better in terms of shoot proliferation, shoot length and number of leaves as compared to other concentrations. For rooting of microshoots, a lower concentration of NAA (0.5 µM) yielded more efficient rooting of micro shoots (17.33 roots per micro shoot). In vitro rooted microshoots were hardened and showed 60% survival rate. The content of gallic acid, chlorogenic acid and 4-hydroxybenzoic acid was higher in the in vivo plant. The amount of ferulic acid was higher in the in vitro raised plant when compared to field grown plant. Furthermore, caffeic acid and p-coumaric acid were higher in the in vitro raised plants as compared to field grown plants. This work will facilitate in conservation of this endangered herb and provide necessary plant materials for various biotechnological and pharmaceutical applications.     

Adenine type and diphenyl urea derived cytokinins improve the postharvest performance of <Emphasis Type="Italic">Iris germanica</Emphasis> L. cut scapes

An experiment was designed to evaluate the effect of various adenine derived cytokinins (kinetin and 6-benzylaminopurine) and diphenyl urea cytokinin (thidiazuron) on the postharvest performance of cut scapes of Iris germanica. Flower scapes were harvested with the oldest bud at ‘1 day before anthesis stage’, brought to laboratory under water, cut to a uniform length of 35 cm, divided into three sets viz., kinetin (KIN), 6-benzyl aminopurine (BAP) and thidiazuron (TDZ). Each set of scapes was treated with a particular cytokinin alone or in combination with 0.1 M sucrose. TDZ was effective than KIN and BAP in improving the postharvest life of the I. germanica scapes by 5.4 days as compared to the control (untreated scapes held in distilled water). This was because of the minimum percentage of bud abortion by TDZ application. Cytokinin application resulted in increased antioxidant activity, higher protein and phenolic content, besides a decrease in specific protease activity and α-amino acids in the tepal tissues. Application of TDZ resulted in the maximum increase in the superoxide dismutase, catalase and ascorbate peroxidase activity in the tepal tissues. The scapes treated with BAP and KIN maintained higher carbohydrate content in the tissue samples as compared to control and TDZ treated scapes. TDZ and BAP application resulted in increased membrane stability because of the decreased lipoxygenase activity which prevented membrane lipid peroxidation. Among the cytokinins tested, TDZ proved to be the promising cytokinin in improving the postharvest performance of beautiful flowers of I. germanica scapes.     

Photosynthetic responses of lettuce to downy mildew infection and cytokinin treatment

Changes in primary metabolism of lettuce, Lactuca sativa L. (cv. Cobham Green), induced by compatible interaction with the biotrophic oomycete pathogen Bremia lactucae Regel (race BL 16), under two intensities of illumination in the presence and absence of exogenous cytokinins were studied by chlorophyll fluorescence imaging. Thirteen days post-inoculation leaf discs infected by B. lactucae exhibited impairments of photosynthesis associated with biotrophic infections, including: reductions in photosynthetic pigment contents and the maximum quantum yield of photosystem II photochemistry (F_V/F_M), inhibition of electron transport (Φ_PSII) and increased non-photochemical chlorophyll fluorescence quenching (NPQ). Detected changes in photosynthetic parameters correlated with the leaf area colonized by the pathogen’s intercellular hyphae. Applications of two cytokinins, benzylaminopurine and meta-topolin, previously shown to suppress B. lactucae sporulation if applied 24 h prior to inoculation at a concentration of 200 μM, retarded the pathogen’s asexual reproduction with no apparent negative effects on the host’s photosynthetic apparatus. However, long-lasting treatment of healthy tissues with this high concentration of exogenous cytokinin led to effects parallel to pathogenesis: reductions in photosynthetic pigment contents accompanied by inhibition of photosystem II photochemistry and electron transport. These effects of both prolonged exposure to cytokinins and the pathogenesis were weaker in discs exposed to the lower photosynthetic photon flux density. The role of cytokinins in plant-biotrophic pathogen interactions and their potential as disease control agents are discussed.     

Photosynthetic and leaf anatomical characteristics of Castanea sativa: a comparison between in vitro and nursery plants

The anatomic and functional leaf characteristics related to photosynthetic performance of Castanea sativa growing in vitro and in nursery were compared. The irradiance saturated photosynthesis in in vitro grown plantlets was significantly lower compared to nursery plants (65 vs. 722 μmol m⁻² s⁻¹). The maximum photosynthetic rate (P_Nmax) was 4.0 and 10.0 μmol(CO₂) m⁻² s⁻¹ in in vitro microshoots and nursery plant leaves, respectively. Carboxylation efficiency (C_E) and electron transport rate (ETR) were three-folds higher in nursery plants than in microshoots. The nonphotochemical quenching (NPQ) was saturated at 80 μmol m⁻² s⁻¹ in microshoots suggesting limited photoprotection by thermal dissipation. The microshoots had wide open, spherical stomata and higher stomatal density than nursery plants and they had almost no epicuticular wax. Consequently, the microshoots had high stomatal conductance and high transpiration rate. These anatomic and functional leaf characteristics are likely major causes of the low survival rates of plantlets after ex vitro transfer.     

Kinetin improves photosynthetic and antioxidant responses of <Emphasis Type="Italic">Nigella sativa</Emphasis> to counteract salt stress

The present study involves analysis of growth, photosynthesis, oxidant (H₂O₂) accumulation, and antioxidant enzyme activities in Nigella sativa L. as affected by foliar kinetin (KIN) application during salt stress. The test plants were treated with 75 or 150 mM NaCl since germination and sprayed with either water or 10 μM KIN in 25 days after emergence. Salt stress, especially at the higher NaCl concentration, was found to induce a substantial decrease in leaf relative water content and subsequently in leaf area and stomatal conductance; chlorophyll content and δ-aminolevulinic acid dehydratase (ALA-D) activity were also affected, resulting in the lower net photosynthetic rate and dry matter production. Moreover, H₂O₂ content increased in the salt-treated plants, concomitant with an increase in superoxide dismutase and peroxidase activities; however, the activity of catalase declined. Meanwhile KIN was found to reduce appreciably the adverse effects of salinity, besides favorably modulating antioxidant enzyme activities and alleviating oxidative stress in the test plants, to result in a higher yield as compared to the untreated stressed plants. Overall, the results indicate an optimization of antioxidant defense mechanisms and physiological processes by KIN and a significant role of exogenous phytohormones in conferring salt tolerance.     

An efficient in vitro regeneration of Ceropegia noorjahaniae: an endemic and critically endangered medicinal herb of the Western Ghats

An efficient protocol was developed for the rapid in vitro multiplication of an endemic and critically endangered medicinal herb, Ceropegia noorjahaniae Ans., via enhanced axillary bud proliferation from nodal explants. The effects of phytohormones [6-benzylaminopurine (BAP), kinetin (Kin) thidiazuron (TDZ), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA)] on in vitro regeneration were investigated. The highest number of shoots (18.3 ± 1.3), maximum shoot length (10.1 ± 0.8 cm) and the highest response of shoot induction (95 %) were recorded on MS medium supplemented with 2.0 mg/l BAP. Rooting was best achieved on half-strength MS medium augmented with IBA (1.0 mg/l). Half-strength MS medium supplemented with BAP (4 mg/l) and sucrose (5 %, w/v) produced an average of 5.6 flower buds per microshoots with highest (90 %) flower bud induction response. The plantlets regenerated in vitro with well-developed shoot and roots were successfully established in pots containing sterile sand and coco peat (1:1) and grown in a greenhouse with 85 % survival rate. The regenerated plants did not show any detectable morphological variation. The developed method can be successfully employed for large-scale multiplication and conservation of C. noorjahaniae.     

Role of polarity in de novo shoot bud initiation from stem disc explants of <Emphasis Type="Italic">Curculigo orchioides</Emphasis> Gaertn. and its encapsulation and storability

The occurrence of strong polarity towards shoot bud induction and the effect of cytokinin(s) on each segment of stem axis, encapsulation and storability of de novo Shoot buds of Curculigo orchioides Gaertn. (Hypoxidaceae) have been documented in the present communication. Maximum number of shoot buds arising de novo from the stem discs (cross section) explanted from proximal end on MS medium fortified with BAP and KIN 1 mg/L each. Stem discs from distal end were less efficient in shoot bud induction. A combination of two cytokinins (BAP and KIN) as a synergistic effect on shoot buds induction from each segment of stem axis. Stem discs in inverted position produced shoot buds from the lower surface, showing strong polarity within the explant. Further, storability and shoot development of sodium alginate encapsulated shoot buds of Curculigo orchioides were tested on half-strength Murashige and Skoog (MS) basal medium fortified with coconut water (10% v/v). The frequency of regeneration from encapsulated shoot buds was affected significantly by concentration of sodium alginate and the duration of exposure to calcium chloride. Shoot buds encapsulated with 2.5% sodium alginate dissolved in MS basal salts solution recorded significantly higher shoot development than other treatments. A relatively short (5 min) incubation with calcium chloride solution provided uniform encapsulation of shoot buds that gave the highest percentage (68%) of shoot development. Encapsulated shoot buds could be stored at 4°C for 50 days without reduction in viability as oppose to non-encapsulated shoot buds, which showed 9.5% viability after 20 days at 4°C. Encapsulated shoot bud developed into normal shoots. Based on the present observations an improved protocol may be developed for the rapid multiplication and conservation of the endangered species—C. orchioides.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Growth,fluorescence, photosynthetic O<Subscript>2</Subscript> production and pigment content of salt adapted cultures of <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The effect of salt concentration (NaCl) on growth, fluorescence, photosynthetic activities and pigment content of the cyanobacterium Arthrospira platensis has been investigated over 15 days. It has been observed that high NaCl concentration induces an increase of the growth, photosynthetic efficiency (α), phycobilin/chlorophyll ratio and a slight decrease of dark respiration and compensation points. Moreover, high NaCl concentration enhances photosystem II (PSII) activity compared to photosystem I (PSI). Results show that the phycobilin-PSII energy transfer compared to the chlorophyll-PSII (F_695,600/F_695,440) increases. However, data obtained about the maximal efficiency of PSII photochemistry are controversial. Indeed, the Fv/Fm ratio decreases in salt adapted cultures, while at the same time the trapping flux per PSII reaction center (TR₀/RC) and the probability of electron transport beyond Q_A (₀) remain unchanged at the level of the donor and the acceptor sites of PSII. This effect can be attributed to the interference of phycobilin fluorescence with Chl a when performing polyphasic transient measurements.     

Herbivore perception decreases photosynthetic carbon assimilation and reduces stomatal conductance by engaging 12‐oxo‐phytodienoic acid,mitogen‐activated protein kinase 4 and cytokinin perception

Herbivory‐induced changes in photosynthesis have been documented in many plant species; however, the complexity of photosynthetic regulation and analysis has thwarted progress in understanding the mechanism involved, particularly those elicited by herbivore‐specific elicitors. Here, we analysed the early photosynthetic gas exchange responses in Nicotiana attenuata plants after wounding and elicitation with Manduca sexta oral secretions and the pathways regulating these responses. Elicitation with M. sexta oral secretions rapidly decreased photosynthetic carbon assimilation (A_C) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and 1‐5 ribulose‐1,5‐bisphosphate turnover. Phytohormone profiling and gas exchange analysis of oral secretion‐elicited transgenic plants altered in phytohormone regulation, biosynthesis and perception, combined with micrografting techniques, revealed that the local photosynthetic responses were mediated by 12‐oxo‐phytodienoic acid, while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signalling mediated by mitogen‐activated protein kinase 4. The analysis also revealed a role for cytokinins interacting with mitogen‐activated protein kinase 4 in CO₂‐mediated stomatal regulation. Hence, oral secretions, while eliciting jasmonic acid‐mediated defence responses, also elicit 12‐oxo‐phytodienoic acid‐mediated changes in stomatal conductance and A_C, an observation illustrating the complexity and economy of the signalling that regulates defence and carbon assimilation pathways in response to herbivore attack.     

Structure of the photosynthetic apparatus of the bean Vicia faba L. when treated with 6-benzylaminopurine

The level of endogenous cytokinins changed with growth and development of faba bean (Vicia faba L.) leaves. Typical of juvenile leaves, amounting to 25% of the final leaf size (Smax) was a low content of these plant hormones. The level of cytokinins increased in growing leaves (50% of Smax) and decreased in the leaves that stopped growing. The content of cytokinins in senescent leaves dropped considerably. Exogenous treatment with 6-benzylaminopurine (BAP) had no effect on the structure of terminal phloem; however, it stimulated elongation of mesophyll cells; increases in the area and thickness of leaf blade, amount of photosynthetic pigments, and assimilation potential; and delayed senescence of leaves and defoliation, thereby increasing biomass of the aboveground plant part. It was inferred that BAP had a potential for induction of photosynthetic apparatus development and increase in the yield of faba bean green mass.     

Direct adventitious shoot bud formation on hypocotyls explants in Millettia pinnata (L.) Panigrahi- a biodiesel producing medicinal tree species

A reproducible protocol developed for in vitro regeneration of Milletia pinnata using hypocotyl segments. Multiple shoots were induced from hypocotyl explants through direct adventitious shoot bud regeneration. The proximal end of hypocotyls was responsive for shoot bud induction. Silver nitrate and adenine sulphate had a positive effect on shoot bud induction and elongation. The maximum response and number of shoot bud produced in media supplemented with 8.88 μM BAP with 108.6 μM adenine sulphate and 11.84 μM silver nitrate. Elongated shoots were harvested and successful rooting of microshoots achieved on MS media supplemented with 9.84 μM IBA, with 81.1 % rooting. Remaining shoot buds sub-cultured for further multiplication and elongation. Each subculture produced eight to nine elongated microshoots up to four subcultures. The rooted microshoots were successfully hardened and transferred to field.     

Rapid micropropagation of mature wild cherry

Explants taken from the mature vigorous tree of wild cherry (Prunus avium L.) were assayed for their organogenic capacity under various phytohormonal treatments. The highest rate of adventitious shoot multiplication was recorded at a combination of 0.5 mg dm⁻³ 6-benzylaminopurine (BAP) and 0.05 mg dm⁻³ thidiazuron (6.83 shoots per explant). No differences in multiplication rates were found among media supplemented with BAP, BAP + α-naphthaleneacetic acid (NAA) or BAP + indole-3-butyric acid (IBA). Shoot elongation was significantly affected by the concentration of BAP, regardless of auxin addition to medium. Up to 73 % of microshoots rooted after using 0.3 mg dm⁻³ IBA, otherwise the adventitious rooting occurred at reasonable frequencies in all auxin treatments. Regenerated plantlets were successfully hardened ex vitro and continued to grow after the transfer to soil. No morphological aberrations were observed in the regenerates.     

A two-step procedure for in vitro multiplication of cloudberry (<Emphasis Type="Italic">Rubus chamaemorus</Emphasis> L.) shoots using bioreactor

Cultures of three cloudberry (Rubus chamaemorus L.) clones collected from natural stands in Newfoundland and Labrador, Canada were established in vitro on a modified cranberry (Vaccinium macrocarpon Ait.) tissue culture medium containing 8.9 μM 6-benzylaminopurine (BAP). Clones were compared for in vitro shoot proliferation on gelled medium supplemented with varying levels of BAP and thidiazuron (TDZ). Addition of 5.8 μM gibberellic acid (GA₃) in 8.9 μM BAP-contained medium improved shoot proliferation. TDZ supported rapid shoot proliferation at low concentration (1.1 μM) but induced 20–30% hyperhydricity in a plastic airlift bioreactor system containing liquid medium. Bioreactor-multiplied hyperhydric shoots were transferred to gelled medium containing 8.9 μM BAP and 5.8 μM GA₃ and produced normal shoots within 4 weeks of culture. Genotypes differed significantly with respect to multiplication rate with ‘C1’ producing the most shoots per explant. Proliferated shoots were rooted on a potting medium with 65–75% of survivability of rooted plants. Present results suggested the possibility of large-scale multiplication of cloudberry shoots in bioreactors.