Response to increasing rates of boron and NaCl on shoot proliferation and chemical composition of in vitro kiwifruit shoot cultures

The in vitro response of kiwifruit (Actinidia deliciosa) to increasing concentrations of boron (B) and NaCl in the culture medium was studied. Kiwifruit shoot cultures were grown in vitro for 12 weeks on an MS medium containing two B concentrations (0.1 and 2 mM) combined with five NaCl concentrations (0, 10, 20, 40 and 80 mM). Kiwifruit produced the longest shoots with 2 mM B when NaCl concentration was 0--20 mM. More shoots were produced with 2 mM B for all NaCl treatments. More shoots were produced with 2 mM B and 10 and 20 mM NaCl. High B concentrations in the culture medium significantly increased shoot proliferation. Explants exhibited a moderate chlorotic appearance with 40 mM NaCl and shoots died with 80 mM NaCl. With 2 mM B, the B concentration of explants was 5--9X greater for the various NaCl treatments compared to the control. Increasing the NaCl concentration from 10 to 80 mM, resulted in higher Na and Cl concentrations in explants for all B treatments, while K and Ca concentrations decreased. Phosphorus concentration in the explants was significantly increased by increasing the NaCl concentration reaching a maximum value at 80 mM NaCl for the two B concentrations.     

Response of the pear rootstock to boron and salinity in vitro

The effects of boron and NaCl induced salinity on growth and mineral composition of the pear (Pyrus communis L.) rootstock OH × F 333 shoots cultured in vitro were investigated. Shoots were grown in vitro for seven weeks on a Murashige and Skoog medium containing two B concentrations (0.1 and 2 mM) combined with five NaCl concentrations (0, 10, 20, 40, and 80 mM). The longest shoots were produced at 0.1 mM B and 80 mM NaCl, but highest number of shoots were produced at 0.1 mM B and 0–20 mM NaCl. Inclusion of 20 and 40 mM NaCl in the culture medium significantly increased fresh mass of cultures compared to 0 mM NaCl for all B concentrations tested. The concentrations of P, K, Ca, Mg, Na, Fe, Mn and Zn of plants were affected by B and NaCl concentration of the medium.     

In vitro plantlet regeneration from cotyledon, hypocotyl and root explants of hybrid seed geranium

In vitro plantlet regeneration systems for the seed geranium (Pelargonium x hortorum Bailey) using cotyledon, hypocotyl and root explants were optimized by studying the influence of seedling age, growth regulators and excision orientation on organogenesis. Indole-3-acetic acid combined with zeatin yielded the highest rate of shoot production on cotyledon explants (0.2–2 shoots per explant). More shoots were produced on explants cut from the most basal region of cotyledons from 2 to 4-day-old seedlings than from older seedlings or more distal cut sites. Hypocotyl explants produced the highest number of shoots, up to 40 shoots per explant, on indole-3-acetic acid (2.8–5.6 mM) + zeatin (4.6 mM) or thidiazuron (4.5 mM). Maximum shoot formation (0.3–1.4 shoots per explant) on root explants occurred when they were cultured on medium containing zeatin. Regenerated shoots rooted best on a basal medium containing no growth regulators. There were substantial differences among cultivars in shoot formation from each of the explant systems.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid - TDZ thidiazuron     

Effect of boron and methionine on growth and ion content in kiwifruit shoots cultured in vitro

The growth of kiwifruit explants was affected by boron (B) and methionine (Meth) in the culture medium. The longest shoots, the greatest number of shoots and the highest amount of fresh mass per explant were produced in Murashige and Skoog medium with 2 mM B and 2 μM Meth. Furthermore, by increasing B concentration in the culture medium from 0 to 2 mM, an increased rate of shoot proliferation was observed for the various Meth concentrations employed.     

A two-step procedure for adventitious shoot regeneration on excised leaves of lowbush blueberry

An efficient system to regenerate shoots on excised leaves of greenhouse-grown wild lowbush blueberry (Vaccinium angustifolium Ait.) was developed in vitro. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, medial, and basal segments of the leaves was tested. Leaf cultures produced multiple buds and shoots with or without an intermediary callus phase on 2.3–4.5 μM TDZ within 6 wk of culture initiation. The greatest shoot regeneration came from young expanding basal leaf segments positioned with the adaxial side touching the culture medium and maintained for 2 wk in darkness. Callus development and shoot regeneration depended not only on the polarity of the explants but also on the genotype of the clone that supplied the explant material. TDZ-initiated cultures were transferred to medium containing 2.3–4.6 μM zeatin and produced usable shoots after one additional subculture. Elongated shoots were dipped in 39.4 mM indole-3-butyric acid powder and planted on a peat:perlite soilless medium at a ratio of 3:2 (v/v), which yielded an 80–90% rooting efficiency. The plantlets were acclimatized and eventually established in the greenhouse with 75–85% survival.     

Optimized Shoot Regeneration System for the Commercial Korean Radish ‘Jin Ju Dae Pyong’

We have investigated the usefulness of hypocotyl (cultured on N1B2 medium) and cotyledon explants (on CR medium: Murashige and Skoog (MS) medium, 3% sucrose, 20 μm benzylaminopurine, pH 5.8) for the regeneration of shoots of the Korean radish ‘Jin Ju Dae Pyong’. The importance of ethylene (indirectly), polyamines and gelling agent were studied in both media. Although the addition of ethylene-inhibitors and silver nitrate to the culture media were beneficial towards shoot regeneration and agar-based treatments (0.8% w/v) being superior in shoot production compared to agarose treatments (0.4% w/v), both explants responded differently in culture. Hypocotyls cultured in the presence of silver nitrate or aminoethoxyvinylglycine (AVG) regenerated significantly (p < 0.05) more shoots compared to N1B2 medium alone; supplementation of 20 μM AVG to N1B2 medium gave optimal shoot production (40% of explants regenerating shoots). The addition of 10 μM AVG to CR medium produced maximum shoot regeneration from cotyledon explants (60% producing shoots). Plants derived from 3-month-old cultures produced greater seed weights, larger leaves and greater genetic variability (50–80% of cells having 20–40 chromosomes) compared to seed-derived (85–90% diploid) and plants from 1-month-old cultures (78–88% diploid). Our results show, that if prolonged culture of explants is avoided, a large number of phenotypically-normal plants can be produced, which in turn, could be utilized in the genetic improvement of radish.     

Physiological and growth changes in micropropagated Citrus macrophylla explants due to salinity

Salinity is one of the major abiotic stresses affecting arable crops worldwide, and is the most stringent factor limiting plant distribution and productivity. In the present study, the possible use of in vitro culture to evaluate the growth and physiological responses to salt-induced stress in cultivated explants of Citrus macrophylla was analyzed. For this purpose, micropropagated adult explants were grown in proliferation and rooting media supplemented with different concentrations of NaCl. All growth parameters were decreased significantly by these NaCl treatments; this was accompanied by visible symptoms of salt injury in the proliferated shoots from 60 mM NaCl and in the rooted shoots from 40 mM NaCl. Malondialdehyde (MDA) increased with increasing salinity in proliferated shoots, indicating a rising degree of membrane damage. The concentration of total chlorophyll significantly decreased in the presence of NaCl, and this effect was more pronounced in the rooted explants. The Na⁺ and Cl⁻ concentrations in the explants increased significantly with the salinity level, but Cl⁻ levels were higher in the proliferated explants than in the rooted explants. For osmotic adjustment, high concentrations of compatible solutes (proline and quaternary ammonium compounds—QAC) accumulated in salt-stressed plants in proliferation, but differences were not observed in rooted explants. In proliferation, proline and QAC were highly correlated with the sodium and chloride concentrations in the explants, indicating a possible role of these compounds in osmotic adjustment. The plant concentrations of NO₃⁻, K⁺, Mg²⁺, Ca⁺ and Fe were also affected by the NaCl concentration of the medium. We suggest that the important deleterious effects in the in vitro explants of Citrus macrophylla grown at increasing NaCl concentrations were due mainly to toxic effects of saline ions, particularly Cl⁻, at the cellular level.     

Direct plant regeneration from cucumber embryonal axis

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 μM BA and 1.59 μM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 μM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 μM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.     

Sucrose level influences micropropagation and gene delivery into leaves from in vitro propagated highbush blueberry shoots

In an effort to increase in vitro blueberry (Vaccinium corymbosum L.) shoot production without negatively impacting subsequent genetic engineering experiments, studies were conducted to examine the effects of sucrose concentration in the propagation medium on shoot proliferation and on the transfer of an intron-containing -glucuronidase (GUS) gene into leaf explants from the propagated shoots. Numbers of axillary shoots >0.5 cm in length did not significantly increase for `Bluecrop' when sucrose levels were increased from 15 mM to either 29, 44 or 58 mM. The number of axillary shoots increased significantly for Duke ' and `Georgiagem' when sucrose concentrations were increased from 15 to 44 mM, and from 15 to 58 mM, respectively. Four-days of cocultivation with Agrobacterium tumefaciens strain EHA105 yielded highest GUS-expressing leaf zones on leaf explants from shoots cultured on either 15 or 29 mM sucrose. The number of GUS-expressing leaf zones was significantly lower on leaf explants derived from shoots grown on 58 mM sucrose than from those grown on 15 mM sucrose for all three cultivars, and was significantly lower on 44 mM compared to 15 mM for cultivars Duke and Georgiagem. These studies indicate shoot pretreatment conditions for optimizing subsequent blueberry genetic engineering experiments. Thus, a blueberry shoot proliferation medium containing 15–29 mM sucrose is recommended for explants later used for genetic transformation.     

Direct shoot organogenesis from <Emphasis Type="Italic">in vitro</Emphasis>-derived mature leaf explants of pistachio

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Pistacia vera L. cv. Siirt. The in vitro procedure involved four steps that included (1) induction of shoot initials from the regenerated mature leaf tissue, (2) regeneration and elongation of shoots from the shoot initials, (3) rooting of the shoots, and (4) acclimatization of the plantlets. The induction of shoot initials was achieved on an agarified Murashige and Skoog (MS) medium with Gamborg vitamins supplemented in different concentrations of benzylaminopurine (BA) and indole-3-acetic acid (IAA). The best medium for shoot induction was a MS medium with 1 mgl⁻¹ IAA and 2 mgl⁻¹ BA. Numerous shoot primordia developed within 2–3 wk on the leaf margin and the midrib region, without any callus phase. In the second step, the shoot clumps were separated from the leaf explants and transferred to a MS medium supplemented with 1 mgl⁻¹ BA, resulting in a differentiation of the shoot initials into well-developed shoots. The elongated shoots (>3 cm long) were rooted on a full-strength MS basal medium supplemented with 2 mgl⁻¹ of indole-3-butyric acid in the third stage. Finally, the rooted plants were transferred to soil with an 80% success rate. This protocol was utilized for the in vitro clonal propagation of this important recalcitrant plant species.     

Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures

Experiments were performed to determine the effects ofcarbon source and concentration on shootmultiplication in shoot cultures of Fagussylvatica (one clone) and F. orientalis (twoclones) and on the induction of adventitious shootbuds from leaf and internode explants of F.orientalis. In general, glucose was the best carbonsource for both axillary branching and adventitiousshoot regeneration. Shoot-tip explants grown on 3–4%glucose medium produced more shoots than those onsucrose or fructose. For maximum shoot length, glucosemedium was best for two of the three clones, and 4%sucrose for the other. The number of shoots was theparameter most influenced by glucose concentration inthe adventitious shoot regeneration experiments, thenumber increasing with sugar concentration. The lowesthyperhydricity rate occurred in the presence ofsucrose in both species. Shoot growth and quality wasnegatively affected by fructose supplied media. Theuse of filter-sterilized rather than autoclavedfructose neither stimulated shoot growth nor reducedthe incidence of hyperhydricity in all three clones.The response of shoot cultures to differentcarbohydrate treatments appears to some extent to begenotype dependent.     

Factors affectingin vitro establishment and shoot proliferation ofRosa hybrida L. andRosa Chinensis minima

Summary The effects of nodal explants collected at different plastochrones, use of various benzyladenine (BA) concentrations, sources of carbohydrates, and phases of the culture medium on shoot establishment and proliferation ofRosa hybrida L. andR. chinensis minima were evaluated. Higher numbers of shoots per explant were obtained fromR. hybrida cv. Carefree Beauty explants proximal to the apical meristem than those from distal nodes. However, proliferating shoots derived from plastochrones proximal to the apical meristem had a lower number of leaves/explant and were shorter than those derived from other distal plastochrones. Although shoot proliferation increased with higher BA concentration in the medium, a concentration of 4.4 μM BA was found optimum for axillary bud-break and shoot development forR. hybrida cvs. Adelaide Hoodless and Cuthert Grant. A higher shoot proliferation rate was observed forR. hybrida cv. Carefree Beauty explants grown on a medium containing 55.5 mM fructose than 58.4 mM sucrose. However, no differences were observed forR. hybrida cv. Cuthert Grant grown on a medium containing either fructose or sucrose. The mean number of shoots/explant was higher forR. chinensis minima cv. Red Sunblaze explants grown on a liquid (4.5) than on a solid medium (1.7) for the first reculture; while no significant differences between the two phases of the medium were observed for the second reculture. However, a higher mean number of shoots/explant was observed on solid-phase (4.0) than liquid-phase medium (3.4) for the third reculture. A higher mean number of leaves/shoot was obtained on a solidified medium rather than liquid medium in the first two consecutive recultures, while no differences were observed for the third reculture. Although a significant effect of BA concentration on mean number of shoots/explant was observed for Red Sunblaze nodal explants, the influence of BA concentration decreased in the two consecutive cultures for both phases of the medium. Hyperhydricity was observed on Red Sunblaze shoots grown on the liquid-phase medium.     

Micropropagation of Camptotheca acuminata decaisne from axillary buds, shoot tips, and seed embryos in a tissue culture system

Summary Micropropagation of the anti-cancer plant Camptotheca acuminata Decaisne from axillary buds and seed embryos was investigated. Axillary buds from greenhouse seedlings required a period of culture in media free of N⁶-benzyladenine (BA) before multiple shoot induction began. Direct induction of multiple shoots on BA-containing medium resulted in high mortality of the axillary buds. Multiple shoot induction from the greenhouse axillary buds was best achieved on B5 with 4.4 μM BA+0.5μM α-naphthaleneacetic acid, forming an average of three 2-mm tall shoots per bud in 8 wk. Elongation of these multiple shoots was successful at a lower BA level (0.22 μM) on B5 medium. Both in vitro and ex vitro rooting of the microcuttings was feasible with indole-3-butyric acid in the culture media, but ex vitro rooting led to high plantlet survival. Seed embryos were not ideal explants for multiple shoot induction. Shoot tips and axillary buds of in vitro-germinated seedlings showed an optimal multiple shoot formation on B5 with 8.9 μM BA, double the optimal BA level for greenhouse axillary buds. Using axillary buds to propagate C. acuminata plants in vitro is feasible for mass propagation of desired clonal lines high in camptothecin concentrations.     

Thidiazuron promotes high frequency regeneration of peanut (Arachis hypogaea) plants in vitro

Multiple shoots were induced on Valenciatype peanut (Arachis hypogaea L.) explants cultured in vitro on a nutrient medium supplemented with thidiazuron. Zygotic embryos excised from mature seeds were germinated on Murashige-Skoog nutrient medium, and the resulting plantlets (8 days-old) were used as a source of explants. When cultured on a nutrient medium with increasing levels of thidiazuron (0.5 to 30 mg/l), expiants from various parts of the peanut plant (except the root) produced multiple shoot primordia which subsequently developed into individual shoots. Hypocotyl and cotyledon explants produced shoots in higher numbers than other explants (20 shoots per hypocotyl explant at all thidiazuron concentrations and 15 shoots per cotyledon explant at 30 mg/l). Shoots rooted normally on a basal Murashige-Skoog medium containing charcoal and developed into healthy and fertile plants when planted in soil.Abbreviations TDZ thidiazuron - MSO Murashige and Skoog (1962) basal medium - BA 6-benzylaminopurine     

Strawberry sepal: Another explant for thidiazuron-induced adventitious shoot regeneration

Summary An efficient system to regenerate shoots on excised sepals (calyx) of greenhouse-grown ‘Bounty’ strawberry (Fragaria x ananassa Duch.) was developed in vitro. Sepal cultures produced multiple buds and shoots without an intermediary callus phase on 2–4 μM 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (thidiazuron, TDZ)-containing shoot induction medium within 4–5 wk of culture initiation. Young expanding sepals with the adaxial side touching the culture medium and maintained for 14 d in darkness produced the best results. In a second experiment, sepals proved more effective than the leaf discs and petiole segments for regenerating shoots. A third experiment compared the effects of six concentrations of two cytokinins (TDZ at 0, 0.5, 2, and 4 μM and zeatin at 2 and 4 μM) for elongation of sepal-derived adventitious shoots. The media containing TDZ generally promoted more callus formation and suppressed shoot elongation. TDZ-initiated cultures transferred into the medium containing 2–4 μM zeatin, produced usable shoots after one additional subculture. Shoots were rooted in vitro in the same medium used for shoot regeneration, but without any growth regulators. When transferred to potting medium, 85–90% of in vitro plantlets survived.     

Direct shoot regeneration from node,internode, hypocotyl and embryo explants of Withania somnifera

In vitro studies were initiated with Withania somnifera (L.) Dun. for rapid micropropagation of selected chemotypes using nodes, internodes, hypocotyls and embryo explants. Direct regeneration of shoot buds was observed in MS basal medium supplemented with various concentrations of either benzyladenine (BA) or thidiazouron (TDZ) depending on the explant. Nodal explants formed multiple shoots both from pre-existing and de novo buds on Murashige and Skoog's medium (MS) containing 0.1–5.0 mg l⁻¹ BA and a ring of de novo shoot buds on MS medium containing 0.2 and 0.3 mg l⁻¹ TDZ. Internodal explants formed shoot buds on MS with 1.0 and 5.0 mg l⁻¹ BA while the hypocotyl explants gave rise to multiple shoots only on MS with 0.5 mg l⁻¹ BA. Isolated embryos gave rise to many shoot buds on MS with 0.2 and 0.3 mg l⁻¹ TDZ. The shoot buds elongated and rooted either on MS medium with 0.01 mg l⁻¹ BA or on half strength MS medium lacking growth regulators, which depended upon the growth regulator used in the shoot bud induction medium. Except for the embryo-derived plantlets, all other plantlets could be acclimatized with 100% success. This revised version was published online in June 2006 with corrections to the Cover Date.     

Thidiazuron-induced high-frequency axillary and adventitious shoot regeneration in Vigna radiata (L.) Wilczek

Summary Prolific shoot regeneration was achieved in mungbean Vigna radiata (L.) Wilczek from 3-d-old in vitro cotyledonary node and hypocotyl explants from seedlings derived from mature seeds on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.9 μM). An initial exposure to TDZ for 20 d and three successive transfers to fresh medium with reduced thidiazuron levels (0.09 μM) resulted in the regeneration of 104 shoots/explant from the cotyledon and 30 shoots/explant from the hypocotyl. Thidiazuron-associated abnormalities such as short compact shoots, fasciation and leaf growth in the form of rosettes were observed in shoots regenerated from hypocotyl explants. Both axillary and adventitious shoot formation from the explants were confirmed by histology. Through repectitive cycles of regeneration in the presence of TDZ, the number of shoots that could be obtained from the two explant classes within 80 d was significantly higher than with previous reports in mungbean     

An efficient protocol for the regeneration of whole plants of chickpea (Cicer arietinum L.) by using axillary meristem explants derived from in vitro-germinated seedlings

Summary An efficient and reproducible protocol for the regeneration of shoots at high frequency was developed by using explants derived from the axillary meristems from the cotyledonary nodes of in vitro-germinated seedlings of chickpea (Cicer arietinum L.). Culture conditions for various stages of adventitious shoot regeneration including the induction, elongation, and rooting of the elongated shoots were optimized. The medium for synchronous induction of multiple shoot buds consisted of Murashige and Skoog basal medium (MS) with low concentrations of thidiazuron (TDZ), 2-isopentenyladenine (2-iP), and kinetin. Exclusion of TDZ and lowering the concentration of 2-iP and kinetin in the elongation medium resulted in faster and enhanced frequency of elongated shoots. Cultivation of the stunted shoots on MS with giberellic acid (GA₃) increased the number of elongated shoots from the responding explants. pH of the medium played a very crucial role in the regeneration of multiple shoot buds from the explants derived from cotyledonary nodes. A novel rooting system was developed by placing the elongated shoot on a filter paper bridge immersed in liquid rooting medium that resulted in rooting frequency of up to 90%. A comprehensive protocol for successful transplantation of the in vitro-produced plants is reported. This method will be very useful for the genetic manipulation of chickpea for its agronomic improvement.     

Micropropagation of kiwifruit using non-axenic shoot tips

Kiwifruit (Actinidia chinensis Planch.) shoot tips were subjected to a standard surface sterilization procedure and cultured on a Murashige and Skoog basal medium in the presence of two surviving bacterial contaminants. The fresh weight increase of the cultures and the number of shoots produced were greater in liquid medium than in medium solidified with 0.4 or 0.8% agar. A greater number of shoots was obtained with 125 ml than with 50, 250, or 500 ml Erlenmeyer flasks. A concentration of 2 mgl^-1 N⁶-benzylaminopurine (BAP) gave a greater increase in fresh weight than either 0 or 4 mgl^-1.Shoots cut from proliferating cultures were dipped in 0.05% indolebutyric acid (IBA) and rooted directly in a peat: vermiculite: perlite mix. Over 93 % of 907 plantlets produced were successfully acclimatized. The productivity of the method was comparable to that reported for the axenic culture of meristems. The contaminants which survived the initial surface sterilization procedure thus presented no major obstacle to the in vitro propagation of kiwifruit.     

Growth and shoot formation in protoplast-derived calli of Brassica oleracea ssp. acephala and ssp. capitata

The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.