Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

Micropropagation of Eucalyptus benthamii to form a clonal micro-garden

Eucalyptus benthamii is an important component of forestry plantations in cold regions, but it is difficult to obtain clonal plants of this species, especially by low rooting. In this study, we developed a method for cloning selected genotypes of E. benthamii using a micropropagation technique, enabling the formation of a clonal micro-garden. Nodal segments from sprouts of mini-stumps in the clonal mini-garden were used as explants. After in vitro establishment of the explants, we tested two selected clones (BP101 and BP118), three culture media (Wood Plant Medium (WPM), Correia and colleagues JADS medium, and Murashige and Skoog medium), and two plant growth regulators (6-benzylaminopurine (BAP) and ??-naphthaleneacetic acid (NAA)) for the multiplication of adventitious buds. Additionally, combinations of two other plant growth regulators (BAP and gibberellic acid (GA₃)) were tested for the elongation of shoots. The in vitro and ex vitro rooting of micro-plantlets prior to acclimatization were compared. The in vitro bud multiplication of E. benthamii depended on the clone, culture medium, and concentration of plant growth regulators. The best results were obtained with WPM supplemented with 0.5?mg?L^?1 BAP and 0.05?mg?L^?1 NAA. The elongation of shoots depended on the clone and plant growth regulator, and the best results were obtained with nutrient medium free of GA₃ and BAP. Histological analysis showed that both in vitro and ex vitro rooting were successful, resulting in normal development of adventitious roots showing a vascular connection with the vascular cambium. The new protocol is efficient for micro-plantlet production of E. benthamii and can be used for the formation of a clonal micro-garden for other Eucalyptus or tree species.     

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.     

Micropropagation of Dendrocalamus asper {Schult. & Schult. F.} Backer ex k. Heyne): an exotic edible bamboo

Effect of season, media type, carbon source, growth regulators and transplanting media on micropropagation of Dendrocalamus asper, an important bamboo species, was examined. The season of explant collection played an important role in axillary bud sprouting and spring (February?CApril) was found to be the best period for explant collection. Among the different media MS was found to be the best for micropropagation. Maximum numbers (4.83/explant) of shoots were initiated in MS?+?15???M BAP. For shoot multiplication, MS medium supplemented with 10???M BAP and 75???M Adenine sulfate was used. BAP was superior to KIN for both explant establishment, as well as, shoot multiplication. Optimal rooting was achieved in shoots cultured on ? strength MS medium supplemented with 5???M each of IBA and NAA. Regenerated plantlets were acclimatized and hardened in green house using dune sand and vermi-compost (3:1) with 92.34% success and transferred to the field with 100% survival rate. In the field, plants supplied with FYM along with urea showed better growth and development. Macroproliferation, plant multiplication by separating the rooted tillers of well established in vitro raised plantlets after 5 to 6?months of growth in the green house could double the multiplication rate. More than 25000 in vitro raised plants were successfully transferred to the field and no morphological variations in growth were observed, thus proving the potential of tissue culture for raising large scale plantations of D. asper.     

Alternating Cytokinins in Multiplication Media Stimulates in Vitro Shoot Growth and Rooting of Eucalyptus globulus Labill.

Shoots of Eucalyptus globulus Labill. cultured on shoot multiplicationmedia containing, on alternate subcultures, 6-benzylaminopurine(BAP) or 6-furfurylaminopurine (kinetin), showed better growththan cultures in which either of the cytokinins was used continuously,or both were used in an equimolar mixture. When BAP was usedcontinuously in the medium (i.e. in every subculture), shootsmultiplied but remained stunted and leaves became red and abscised.Kinetin or 6-dimethylallyaminopurine (2iP) used continuouslyin the medium induced very low multiplication but the shootsdid not become red nor did the leaves abscise. Shoots takenfrom multiplication medium containing BAP and placed on rootingmedium with 10 µM indole butyric acid (IBA) produced fewroots and often died while on the rooting medium. In contrast,shoots from the multiplication medium containing kinetin producedmore roots and remained healthy during the passage on the rootingmedium.Copyright 1994, 1999 Academic Press Eucalyptus globulus, Tasmanian bluegum, cytokinins, micropropagation, in vitro rooting     

Micropropagation with a novel pattern of adventitious rooting in American sweetgum (Liquidambar styraciflua L.)

Liquidambar styraciflua L. has great potential not only as an ornamental, but also for its commercial importance in pulp and paper production or biomass energy. This study was designed to evaluate the influence of plant growth regulators on adventitious shoot multiplication from shoot tips, and in vitro adventitious rooting. The morphogenic capacity of intact leaves grown in vitro was also assayed for adventitious shoot formation and aerial root development. The highest shoot multiplication rate of 5.9 shoots per explant was achieved with 0.7 mg l⁻¹ 6-benzylaminopurine plus 0.01 mg l⁻¹ indole-3-butyric acid. Thidiazuron, alone or in combination with 6-benzylaminopurine, did not significantly support higher shoot multiplication rates. The organogenic ability of the in vitro grown leaves was significantly lower and slower in comparison with shoot tips. Microshoots rooted readily after transfer to a half-strength woody plant medium supplemented with 0.5–0.7 mg l⁻¹ 1-naphthaleneacetic acid, and were then successfully acclimatised to an ex vitro environment. A novel pattern of adventitious rooting was observed from the aerial parts of microshoots which were not in contact with the medium, including the parenchyma cells of the leaf blades as well as stem nodes and internodes. The regenerated plants established in soil did not show any detectable morphological variation.     

Tissue culture independent transformation for Corchorus olitorius

An efficient microprogation protocol has been developed for Dendrobium densiflorum Lindl. ex Wall., a traditional medicinal plant, through protocorm-like bodies (PLBs) from nodal stem segments using 6-benzylamino-purine (BAP) and the lanthanoid neodymium. The highest percentage of explants producing PLBs (72%), with an average of 15 PLBs per explant, was induced by culturing stem segments on Murashige and Skoog (MS) medium supplemented with 5.0 mg l⁻¹ BAP. The newly formed PLBs proliferated well on the basal MS medium and completely converted into shoots on MS medium containing 2.0 mg l⁻¹ BAP. Shoots produced an average of 22 roots per plantlet when cultured on MS medium supplemented with 2.0 mg l⁻¹ neodymium nitrate. Healthy plantlets with well-developed roots were successfully acclimatized. The obtained result suggests that the lanthanoids can be used to effectively initiate rooting in the micropropagation and conservation of D. densiflorum.     

Micropropagation of apple — A review

Micropropagation of apple has played an important role in the production of healthy, disease-free plants and in the rapid multiplication of scions and rootstocks with desirable traits. During the last few decades, in apple, many reliable methods have been developed for both rootstocks and scions from a practical, commercial point of view. Successful micropropagation of apple using pre-existing meristems (culture of apical buds or nodal segments) is influenced by several internal and external factors including ex vitro (e.g. genotype and physiological state) and in vitro conditions (e.g., media constituents and light). Specific requirements during stages of micropropagation, such as the establishment of in vitro cultures, shoot multiplication, rooting of microshoots and acclimatization are summarized in this review. New approaches for increasing shoot multiplication and rooting for apple and current use of micropropagated plantlets as tools in basic and applied research are also discussed.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and axillary shoot propagation of <Emphasis Type="Italic">Centaurea zeybekii</Emphasis>

In vitro culture is an important aid for ex situ conservation of rare, endemic or threatened plants. In this work, we establish an efficient method for the seed germination, seedling development, and axillary shoot propagation of Centaurea zeybekii Wagenitz. The seeds, collected from a wild population, were surface sterilised and cultured on various in vitro germination media. The effects of photoperiod and temperature on seed germination were also investigated. Germinations were obtained after 6 weeks in culture and the radicle emergence was evaluated as a main indicator. A high frequency of germination was obtained on distilled water supplemented with vitamines and 1 mg/L GA₃. Although the seed germination frequencies were not affected by photoperiod, the highest germination frequency was obtained at 24 ± 2°C. A high frequency of axillary shoot proliferation was produced on MS medium supplemented with 1 mg/L BA. Then, the axillary shoots were separated and transferred to MS medium with or without plant growth regulators for rooting. Rhizogenezis was promoted after 6 weeks only in MS and 1/2 MS media containing 0.5 mg/L IBA. The rooting process was very slow and the percentage of shoot rooting was also very low (15%). The present study not only enables reinforcement of wild plant populations using ex situ growth of individuals, but it also helps to large number of aseptic seedling to use it in clonaly micropropagation studies.     

Micropropagation and experimental field cultivation of Pulsatilla turczaninovii Kryl. et Serg. (Ranunculaceae)

Pulsatilla turczaninovii is an important medicinal plant, valued for high ornamental value of melliferous flowers. We assessed the efficiency of reproduction under in vitro conditions and the ex situ growth capacity of this important representative of the world flora. The seed germination percentage was assessed, followed by determination of micropropagation rate and rooting efficiency. Then, the possibility of plant development in three consecutive growing seasons was assessed. The in vitro germination percentage was approximately 55%. The highest multiplication coefficient, amounting to 5.17, was obtained on modified MS medium supplemented with 2.5 mg L^?1 2iP and 1.0 mg L^?1 IAA. Our study provided unique insight on biochemical background of root regeneration in P. turczaninovii. In comparison with standard auxin-supplemented rooting medium, the treatment with 1.0 mg L^?1 level of ethylene precursor ACC elevated rooting by about 20%. The total content of soluble sugars was proved to be biomarker of rhizogenesis in the studied species. Their concentration was positively correlated with rooting efficiency, while a level of phenolic was positively correlated with the length of regenerated roots, and their number per rosette. The cultivation of the acclimatized material was successfully carried out and was evaluated over three subsequent years. In the third year of cultivation, the plants entered the stage of generative development and most of them bloomed profusely.

     

An improved micropropagation of <Emphasis Type="Italic">Eclipta alba</Emphasis> by in vitro priming with chlorocholine chloride

An efficient method of micropropagation for Eclipta alba from young nodal axils of shoot tip explants has been developed by giving special attention to ‘priming’ in vitro plantlets in view of increasing their hardening ability after transplantation ex vitro. Among 3 cytokinins—BAP, kinetin and TDZ, BAP was found most effective in inducing and proliferating adventitious shoots. The highest frequency of responding explants (100%) and maximum number of shoots (23.0) per explant were obtained after 60 days culture on MS medium containing 8.8 μM BAP. Cent percent shoots developed roots directly from shoot base when transferred to growth regulator-free MS medium. For priming E. alba microshoots, 6.3 μM of chlorocholine chloride (CCC) was found most effective. The major changes observed in 30 days old treated shoots were, production of increased number of root, elevation of chlorophyll level in leaves and increase in plant biomass. Furthermore, arrested undesirable shoot elongation made the plants sturdier and more suitable for acclimatization. The primed micropropagated E. alba plants were healthy and survived by higher frequency (100%) in soil in comparison to the non-treated plants (84% survival).     

Beneficial use of lignosulfonates in in vitro plant cultures: stimulation of growth,of multiplication and of rooting

Lignosulfonates (LIGNs), low-cost by-products from the paper industry, are already commercialized as fertilizers; they stimulate both vegetative and reproductive growths and fructification. LIGNs have been tested in in vitro cultures and here too, they improve shoot growth and vigor, and rooting of various plant materials. This study aimed at to extend the in vitro application of LIGNs at different developmental stages in order to increase the productivity of systems generating vitroplants. The present results showed the beneficial effects of various LIGN applications on growth of a tropical orchid, Phalaenopsis, multiplication of Saintpaulia ionantha and rooting of poplar and Sequoiadendron sempervirens shoot cuttings. One of the most interesting observations was the stimulating effect of Ca-chelated LIGN on growth of Phalaenopsis and on rooting of Sequoiadendron. The significant and reproducible effects of LIGNs at several steps of micropropagation of different plant materials represent a potential tool to improve quality without embarrassing side-effects.     

Mother plant age and seasonal influence on in vitro propagation of <Emphasis Type="Italic">Quercus euboica</Emphasis> Pap., an endemic,rare and endangered oak species of Greece

A micropropagation method for Quercus euboica Pap. was developed. Nodal explants from seedlings gave higher multiplication rates than explants from adult plants. Cultures initiated at the beginning of May produced the highest percentage of shoot forming explants and multiplication rate. Woody Plant Medium (WPM) salts, with 100 mg l⁻¹ myoinositol, 1 mg l⁻¹ thiamine, 0.5 mg l⁻¹ pyridoxine, 0.5 mg l⁻¹ nicotinic acid and 3% sucrose was used as basal medium and several cytokinins at various concentrations were evaluated for their effect on shoot multiplication. The highest shoot multiplication rate was obtained with 4.44 μΜ BA. IBA at 9.84 μΜ in the culture medium during the first week of culture, and if followed by culture in hormone-free medium, gave the best rooting results. Darkness at the beginning of the rooting period did not improve rooting. The use of plastic wrap as a cover material of the culture vessels enhanced rooting percentage and root number. Plantlets acclimatized ex vitro in soil from the natural environment of the species survived at a higher percentage (up to 93%) and had more vigorous growth than plantlets grown in a compost–perlite (2:1 v/v) medium (up to 36%).     

Micropropagation of sweet viburnum (<Emphasis Type="Italic">Viburnum odoratissimum</Emphasis>)

A micropropagation protocol for shoot culture of sweet viburnum (Viburnum odoratissimum) is described. Nodal explants, initially established on MS medium, were transferred to WPM supplemented with combinations of BA and GA₃. Maximum shoot multiplication was observed on explants cultured on medium supplemented with BA concentration higher than 1.1 μM, and 14 μM GA₃. Although Stage II medium supplemented with BA concentration higher than 1.1 μM resulted in increased shoot multiplication, it also caused a decrease in shoot length. A negative carry over effect of GA₃ on rooting was observed in subsequent Stage III cultures. The presence of GA₃ in Stage II medium promoted shoot elongation, but it also caused a decrease in microcutting rooting. For this reason, 0.5 μM BA and 14 μM GA₃ were selected for optimum Stage II shoot multiplication. Although 100% microcuttings formed roots when cultured on medium containing 6.0 μM NAA, significant callus formation was observed and ex vitro survival rate was low (49%). Rooting was achieved after 3 weeks with 82% of microcuttings on medium supplemented with 3 μM IBA. The survival rate of plantlets under ex vitro conditions was 100% after 3 weeks. Plants looked healthy with no visually detectable phenotypic variation based on observation of about 30 plants.     

An improved micropropagation of Terminalia bellirica from nodal explants of mature tree

An efficient and improved in vitro propagation method has been developed for Terminalia bellirica, a medicinally important tree from nodal explants of 10-year-old mature tree. Shoot multiplication was influenced not only by cytokinin types, their concentrations and their interaction with auxin but also by successive transfer of mother explants for different passages, subculture of excised shoots on fresh medium and different medium composition. MS medium containing 2.22 μM BAP was found to be the best for shoot multiplication in a single step. After excision of newly formed shoots, mother explants successively transferred to the same medium produced maximum shoots per explant after IV passage. Further enhancement in morphogenetic response occurred when excised shoot clumps (2–3 shoots) were subcultured on MS medium supplemented with 2.22 μM BAP, 1.16 μM Kn and 0.57 μM IAA. Half-strength MS medium supplemented with 24.60 μM IBA and 100 mg l⁻¹ AC was most effective for rooting of the shoots. To reduce labor, cost and time, an experiment on ex vitro rooting was also carried out and it was observed that highest percent shoots rooted ex vitro when treated with 2,460 μM IBA for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. In comparison to plantlets developed from in vitro rooted, percent survival of plants those rooted ex vitro was significantly higher. Use of ex vitro rooting technique for plant production serves as a more economical option; therefore, present method can be used for large-scale commercial production of this medicinally important tree.     

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.     

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.     

In vitro micropropagation of Basella rubra L. through proliferation of axillary shoots

In vitro micropropagation protocol for Basella rubra regeneration was tried through proliferation of axillary shoots of the potted mature plant. The improved seed germination (70%) was recorded upon 2% urea treatment. The nodal shoot segments from matured potted plant were used to initiate the multiple shoot proliferation. The shoot segments exhibited 70% shoot initiation when cultured on Murashige and Skoog (MS) medium supplemented with Indole-3-acetic acid (IAA)?+?N₆ – Benzylaminopurine (BAP) (0.25?+?2.0 mg/L) and BAP?+?Kinetin (Kin) (2.0?+?0.5 mg/L) respectively. Multiple shoots (5–6) were obtained on MS medium supplemented with BAP?+?Kin and IAA?+?BAP respectively. When compared with silver nitrate (AgNO₃) (2–40 µM) and activated charcoal (AC) (0.1–1.0%), the MS medium devoid of any plant growth regulator showed good number of shoots (5.48?±?2.42), elongation (15.64?±?2.42 cm) and root length (14.52?±?2.78 cm). Upon transferring of regenerated microshoots to MS medium, simultaneous elongation of shoots with more shoot number, shoot length and rooting was achieved during four subcultures that carried out at 6 weeks’ interval. The regenerated in vitro shoots showed 100% rooting in MS medium and also in MS medium supplemented with 0.1–1.0% AC. Hundred percent survival of micropropagated shoots well rooted was established successfully under greenhouse condition and the plants were subsequently acclimatized and transferred to the field conditions wherein 90% success rate was noted.

     

In vitro regeneration of Echinacea purpurea from leaf explants

Efficient plant regeneration was achieved via organogenesis from callus cultures derived from leaf tissue of Echinacea purpurea. Proliferating shoot cultures were obtained by placing leaf explants on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) and naphthaleneacetic acid (NAA) combinations. MS medium supplemented with BAP (4.44 M) and NAA (0.054 M) was the most effective, providing high shoot regeneration frequencies (100%) associated with a high number of shoots per explant (7.7 shoots/explant). Plantlets were rooted on MS medium alone or in combination with different concentrations of indole-3-butyric acid (IBA), and high rooting and survival was achieved using MS media without plant growth regulators (PGR). All plantlets survived acclimatization producing healthy plants in the greenhouse. This study demonstrated that adventitious shoot regeneration of E. purpurea from leaf explants can be a useful method for the multiplication of this important medicinal plant.     

Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Benzyladenine (BA) is the only cytokinin to effectively induce shoot multiplication in vitro between genotypes of the important dune grass species Uniola paniculata (sea oats). However, a significant genotype-specific negative carryover effect of BA on ex vitro acclimatization has been observed. In the present study, the effects of multiplication media supplemented with meta-topolin (mT), a BA-analog, BA or no plant growth regulator, were compared on in vitro multiplication, rooting and ex vitro acclimatization using easy- and difficult-to-acclimatize sea oats genotypes. Both genotypes exhibited similar in vitro shoot dry weight, number of harvestable shoots and percent rooting when cultured under standard conditions (with 2.2 μM BA) or with an equimolar concentration of mT. In addition, both genotypes exhibited similar ex vitro leaf length and shoot production under these two culture conditions. However, ex vitro acclimatization of rooted microcuttings of the difficult-to-acclimatize genotype significantly increased when produced on shoot multiplication medium containing mT rather than BA. Meta-topolin concentrations 10 μM or greater were inhibitory to in vitro rooting and acclimatization ex vitro of both genotypes. Nevertheless, survival of the difficult-to-acclimatize genotype was significantly greater when cultured in the presence of 2.2 μM–30 μM mT, compared to 2.2 μM BA. Therefore, a potential solution to overcome the detrimental BA carryover effect on ex vitro survival in sea oats is the substitution of BA with 2.2 μM mT for Stage II shoot multiplication. Use of mT may provide an efficient method to ensure in vitro propagation of a large number of diverse sea oats genotypes for dune restoration.