Improved Micropropagation Protocol Research Articles

An improved micropropagation protocol for the ex situ conservation of Thlaspi cariense A. Carlström (Brassicaceae): an endangered Turkish endemic plant species

An improved micropropagation protocol for the ex situ conservation of Thlaspi cariense A. Carlström (Brassicaceae): an endangered Turkish endemic plant species

Comparative study of micropropagated plants of Grand Naine banana during in vitro regeneration and ex vitro acclimatization

An improved micropropagation protocol was established from field grown sword suckers of Banana Grand Naine (Musa acuminata AAA) by evaluating the mircopropagules combating reactive oxygen species (ROS) during acclimatization. Optimization of surface sterilization of sprouted apical meristematic buds and pre-culture on Shoot Bud Induction Medium [MS + 4.0 mg l −1 BA under dark conditions for 3 weeks] resulted ideal number of multiple shoot clusters (12.20 ± 0.30). Subculture of these shoot clusters on Shoot Bud Multiplication Medium (SBMM) fortified with 2 mg l −1 BA and 20 mg l −1 AdS induced highest shoot buds (15.00 ± 0.75) in 3 weeks and showed consistent till seven passages. The individual in vitro shoots upon transferring onto MS + 2 mg l −1 KN +1.0 mg l −1 IBA + activated charcoal (3 g l −1) resulted in elongated shoots and in vitro rooting after 3 weeks at a photoperiod of 16 h light and 8 h dark conditions. Evaluation of antioxidant defense status in micropropagated plantlets during acclimatization revealed altered levels of MDA, chlorophyll, catalase, ascorbate peroxidase and glutathione reductase. Our study throws an insight into decisive/differential role of cytokinins (Kinetin) in combating oxidative stress during acclimatization of in vitro regenerated plantlets. The present investigation facilitates the massive propagation (90% survival rate) of healthy and quality in vitro plantlets of banana.

Development of an Improved Micropropagation Protocol for Red-Fleshed Pitaya ‘Da Hong’ with and without Activated Charcoal and Plant Growth Regulator Combinations

Micropropagation protocols for red-fleshed Hylocereus species (Cactaceae) have been developed; however, these methods prolong the sprout duration from areoles and produce irregular micro-propagules in ‘Da Hong’ pitaya. Thus, the present study aimed to establish an improved micropropagation protocol for this cultivar. Shoot regeneration and root induction of self-pollinating seedling segments were evaluated in response to combinations of activated charcoal (AC; 200 mg/L), α-naphthaleneacetic acid (NAA; 0.05, 0.10, and 0.20 mg/L), and 6-benzylaminopurine (BAP; 1.00, 2.00, and 4.00 mg/L). The correlations among plantlet growth characteristics and plantlet survival rate after transplantation under field conditions were calculated. Increasing the NAA concentration increased the number of roots but reduced root length. The addition of AC enhanced shoot length and prevented the regeneration of dried-out, clustered, and abnormal shoots. Plantlets treated with 200 mg/L AC and 0.10 mg/L NAA produced the highest number of shoots, i.e., 4.1 shoots, which however, were shorter and lighter than those cultured with AC alone. Plantlets grown on medium supplemented with BAP showed no advantage in shoot number, shoot weight, plantlet surface area, or plantlet volume. The weight and shoot surface area of plantlets were strongly correlated. All plantlets grew well at 4 weeks post-transplantation. Overall, these results support this improved micropropagation method to regenerate robust ex vitro plantlets.

Improved Micropropagation Protocol and Molecular Marker Based Genetic Stability Assessment of Black Pepper (Piper nigrum L.)

Black pepper (Piper nigrum L.) is a highly commercial and valuable spice crop in the world, plants of which have originated from Western Ghats region of southern India. An efficient and reliable micropropagation protocol is reported in this paper, which can be utilized for multiplication of elite genotypes of black pepper. Shoot regeneration was induced on Murashige and Skoog (MS) and Woody Plant Media (WPM) media in 24 combinations with plant growth regulators. Explants cultured in MS media supplemented with 0.5 mg L-1 BAP formed 6.5 nodes per plant with longest shoots (10.42 cm) after 12 wks. Regenerated shoots were rooted on MS media supplemented with 2.0 mg L-1 IBA, yielding about 15 roots per plant with an average root length of 3.40 cm within 4 wks. The hardening of the shoots under a mist chamber conditions (25±2 °C, 70-80% RH) yielded 100% survival after acclimatization. Genetic stability analysis of the micropropagated and mother plants was done using 48 ISSR markers. The plants found to be genetically stable when compared with mother plants. A ratio of 1:6.5 numbers of nodes is obtained on the MS medium supplemented with 0.5 mgL-1 of BAP within 3 months. This research presents an improved and efficient micropropagation protocol with good rate of multiplication of the genetically stable plants that are easily acclimatized to the field conditions.

Improved micropropagation protocol for pear rootstocks of the OH×F series and a new selection

Improved micropropagation protocol for pear rootstocks of the OH×F series and a new selection

Improved protocol for micropropagation of genetically uniform plants of commercially important cardamom (Elettaria cardamomum Maton)

Cardamom (Elettaria cardamomum Maton) is a highly valuable commercial spice crop, originating from the Western Ghats of southern India. It has importance both as a spice and for its medicinal properties. An efficient and reliable micropropagation protocol using shoot tips as explants is presented which can be utilized for development of elite planting material. Growth patterns of cardamom shoots were analyzed on 45 media treatments and best shoot proliferation was recorded on Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzylaminopurine (BAP) and 2.32 μM kinetin (Kn) with 5.83 shoots regenerated per explant. The longest shoots (6 cm) were observed on 0.44 μM BAP and 2.32 μM Kn containing medium. Regenerated shoots rooted well on full strength MS basal medium, generating 3.50 roots per explant with an average root length of 4.33 cm within 4 wk. Successful hardening with a field survival percentage of 80% was achieved after acclimatization of well-developed plantlets. Genetic stability of the micropropagated plants was confirmed using 13 ISSR markers. This study reports an improved micropropagation protocol for cardamom with an enhanced rate of shoot multiplication leading to the production of healthy shoots which can be easily rooted and successfully transferred to the field.

Analysis of rhizome colour content, bioactive compound profiling and ex-situ conservation of turmeric genotypes (Curcuma longa L.) from sub-Himalayan terai region of India

Turmeric (Curcuma longa L.), the golden spice of India, offers a myriad of health benefits primarily due to the presence of polyphenolic curcuminoid pigments. It is believed that the dark orange colour of turmeric rhizome has wide-spread health benefits; but no reliable evidential proof for this is available till date. It is crucial to discern the association of rhizome colour with health benefits. It is also of prime importance to investigate whether the curcuminoids present in the rhizomes are the sole bioactive compounds contributing to its medicinal properties. In the present study, forty five turmeric genotypes were collected from the sub-Himalayan terai region of India which differed in their rhizome colour. Here, the Turmeric Redness Index (TRI) in terms of rhizome colour content (a*/b*) of the genotypes revealed strong correlation with curcuminoid content (+0.93), total phenol content (+0.76), total flavonoids content (+0.82), iron content (+0.55) and antioxidant activity (–0.90 and –0.92 for DPPH and ABTS assays and +0.82 and +0.95 for TAC and CUPRAC assays, respectively, with p < 0.001). Among the studied genotypes, TCP 2 (Turmeric Collection Pundibari 2) having dark orange coloured rhizome turned out to be a superior genotype in terms of its antioxidant potential, curcuminoid and iron content. Quantification of phenolic and flavonoid compounds of TCP 2 revealed that other than the three isoforms of curcuminoids, different therapeutically important bioactive compounds like p-coumaric acid (162.46 mg/kg), catechin (107.67 mg/kg), sinapic acid (417.36 mg/kg) and flavonoid like quercetin (2746.21 mg/kg) are also present in the rhizome, suggesting that along with curcuminoid these compounds also contribute towards its antioxidant potential. The GC–MS analysis of the essential oil of TCP 2 revealed the presence of thirty three volatile compounds with significantly high ar-turmeron (28.57 %), curlone (10.05 %), eucalyptol (10.13 %) and chemigran (13 %). A rapid and improved micro-propagation protocol was also developed for TCP 2 using single rhizome bud sprout as explants. A media composition of MS with 6 ppm BAP and 2 ppm NAA was found to be the best for rapid and efficient micro shoot development.

The effect of silver nitrate on micropropagation of Moringa oleifera Lam. an important vegetable crop of tropics with substantial nutritional value.

An improved micropropagation protocol facilitating continuous multiplication of elite germplasm of Moringa oleifera has been developed. Initial culture of nodal explant in MS medium supplemented with 2.5µM BA resulted in the formation of 12.5 shoots per explant with high frequency of leaf fall (84.3%). To confirm whether the leaf fall is due to accumulation of ethylene in the culture vessel, effect of ethylene releasing agent CEPA in the medium was tested. In order to reduce leaf fall and improve multiplication, varying concentration of anti-ethylene agent, AgNO3 was incorporated in the medium. Addition of 2.5μM AgNO3 in combination with 2.5μM BA produced maximum number of shoots (17.6) including shoots originated from the base of the explant and shoots from the axillary buds of the primary shoots, where significant reduction in leaf fall (20.6%) was noticed. This enabled sustained multiplication of M. oleifera through continuous subculture without adversely affecting shoot number or shoot quality in terms of shoot length. Microshoots obtained from fourth subculture onwards were used for ex vitro rooting and found that by treating 50µM NAA for 30s, maximum numbers of microshoots (83.3%) were rooted. Rooted plants were acclimatized, survived and were successfully transferred to field. Genetic fidelity analysis using 10 ISSR primers revealed more than 95% monomorphic bands among plants raised in MS medium containing low concentration (2.5µM) of AgNO3 and BA (2.5µM). The addition of AgNO3 in the medium sustained in vitro growth and effectively prevented leaf fall compared to control, thus demonstrating efficient micropropagation of M. oleifera.

Micropropagation of Little Bluestem (Schizachyrium scoparium L.)

Native grasses are increasingly used in the landscape. Little bluestem (Schizachyrium scoparium L.), a perennial bunchgrass native to most of the United States, has ornamental traits, such as variation in leaf color, differences in growth morphology, and attractive seed heads. Traditionally, cultivars of little bluestem are propagated by division, which limits the production of new plants. Our objective in this study was to develop an improved micropropagation protocol for little bluestem that would produce true-to-type plants. In 2016, we cultured immature inflorescences of eight genotypes of little bluestem on Murashige and Skoog (MS) medium with four combinations of kinetin (1.0 or 2.0 mg·L−1) and 2,4-D (0.5 or 1.0 mg·L−1) under three levels of light (dark, semilight, full light) to initiate callus. Cultures were evaluated 30 days after initiation and those that had initiated callus were subcultured. Media for subculturing and rooting contained either 0.1 mg·L−1 or no 1-Naphthaleneacetic acid (NAA). Light level had no effect on callus initiation. Initiation media with 1.0 mg·L−1 kinetin and either level of 2,4-D induced callus at almost twice the rate of media with 2.0 mg·L−1 kinetin, and cultures initiated on those media also produced almost twice the number of rooted plants over all genotypes. Genotype affected the number of rooted plants produced. The addition of NAA to medium for subculturing and rooting did not increase the number of rooted plants. In 2017, we cultured immature inflorescences of four genotypes of little bluestem on MS medium with 0.5 mg·L−1 2,4-D and either 1.0 mg·L−1 kinetin or 6-benzylaminopurine (BAP) under full light. Cultures were evaluated 30 days after initiation. Cultures that had initiated callus were subcultured onto MS medium with the same growth regulators as the initiation medium but without 2,4-D. Cultures were cycled between subculture medium with growth regulator and subculture medium with no additional growth regulator until rooted. Cultures initiated and subcultured on medium with BAP initiated two to three times more callus than those on kinetin and produced twice as many rooted plants. Our recommendation for rapid micropropagation of little bluestem is to initiate cultures on MS medium with 1.0 mg·L−1 BAP and 0.5 mg·L−1 2,4-D. After callus initiation, cultures should be subcultured to medium with BAP but no 2,4-D, alternating with medium with no additional growth regulators, until rooted.

An improved micropropagation and ex vitro rooting of a commercially important crop Henna (Lawsonia inermis L.).

An improved micropropagation protocol has been developed for a cosmetically important, dye yielding crop, henna (Lawsonia inermis). Quality of henna product is governed by naphthoquinone based pigment lawsone, thus in vitro multiplication of superior healthy plant to achieve enhanced productivity in terms of dye content and biomass deserve due attention. In the present study, nodal explants collected from an elite plant screened on the basis of superiority in lawsone content was cultured on MS medium supplemented with 0.5μM benzyl adenine (BA) gave significantly (p < 0.05) high number of shoots (24.33). The explants placed on MS medium augmented with 0.5μM BA and 2-isopentenyladenine (2-iP) resulted in the formation of maximum number of shoots (43.67) and was elongated (12.57cm) within 4weeks of culture period. Enhanced axillary bud proliferation and production of mass number of micro shoots was achieved by the continuous subculture in MS medium containing 0.5μM BA and 2-iP. In vitro raised micro shoots were dipped in 0.44mM NAA for 5min followed by planting in polyethylene pots containing a soil: vermiculite (1:1 v/v) mixture produced rooted plantlets (100%). Different auxin types and its concentrations had significant role rooting of L. inermis. Rooting response of various size shoots of L. inermis treated with 0.44mM NAA showed 100% rooting in 4.1-5cm size class shoots. After two months of potting, survived (95%) plants were successfully transferred to medicinal plant garden of the Department. The lawsone content of one-year-old micropropagated plants (23.04mg/gdw) growing in normal environmental conditions and elite mother plant (22.84mg/gdw) was almost similar. Through the present study, efficient cloning of superior germplasm of L. inermis was established.

Highly Efficient Proliferation and Regeneration of Protocorm-like Bodies (PLBS) of the Threatened Orchid, Phalaenopsis bellina

Phalaenopsis bellina is an important indigenous fragrant orchid threatened with extinction. In this study, we evaluated the effect of medium strength, sucrose, nitrogen (NH4NO3) and potato extract on proliferation of P. bellina protocorm-like bodies (PLBs) to improve micropropagation in this species. Optimal treatment for PLBs proliferation rate with an average fresh weight (FW) of 0.97±0.16 g was obtained through culturing on half strength (½) MS medium containing 20 g/L sucrose, 15 mM NH4NO3 and 20% w/v potato extract supplemented with 0.8 μM 2,4 dichlorophenoxyacetic acid (2,4-D). The optimal treatment produced large, healthy and greenish PLBs with reduction in the occurrence of culture browning. In contrast, treatments with high potato extract (>20% w/v) or NH4NO3 (>30 mM) concentrations tend to have inhibitory effect and resulted in low PLBs proliferation rate, with an average FW of 0.77±0.15 g and 0.69±0.15 g, respectively. Plant regeneration of PLBs was achieved on plant growth regulator (PGR)-free ½ MS medium. In total, 60 healthy PLBs from the optimal treatment were successfully regenerated, acclimatized with 100% survival percentage and grew well in a mixture of soil, sand and vermicompost (8:4:2 (w/w/w)). With the optimal treatment, PLBs proliferation rate was enhanced by 27.63%. Our findings offer an improved micropropagation protocol of the endangered P. bellina for conservation and commercial production.

An improved micropropagation protocol for the recalcitrant plant Capsicum – a study with ten cultivars of Capsicum spp. (C. annuum, C. chinense, and C. frutescens) collected from diverse geographical regions of India and Mexico

ABSTRACTCapsicum spp. is a commercially important crop of the Solanaceae family, well-known for its multipurpose use as a vegetable, spice, medicinal and ornamental plants. The genus Capsicum is a recalcitrant species in terms of in vitro morphogenesis and plant regeneration. An efficient method was developed for multiple shoot regeneration in 10 cultivars of Capsicum collected from diverse geographical regions of India and Mexico. Seeds germinated in vitro on a half-strength Murashige and Skoog (MS) medium supplemented with 3.0 % sucrose. Nodes of the in vitro germinated seedlings were used as explant for micropropagation. The combination of the 6-benzylaminopurine, indole-3-acetic acid, and spermidine was found to be the best for multiple shoot induction. However, the optimum responcse varied accompanied by different cultivers with maximum 8.9 ± 0.52 (Capsi-10) to 15.3 ± 0.69 (Capsi-5) multiple shoot per explant. Depending on the cultivar, multiplied shoots were successfully rooted with maximum 18.4 ± 0.20 (highest for Capsi-9) to 36.8 ± 0.29 (highest for Capsi-5) roots per shoot on half-strength MS medium supplemented with 2.0 mg l−1 indole-3-butyric acid, 1.0 mg l−1 α-naphthalene acetic acid, and 1.5 mM spermidine. Finally, the micropropagated plantlets were acclimatized with 40.0–86.7 % survival rate, depending on different cultivars.

Micropropagation of &amp;lt;i&amp;gt;Curcuma Sp&amp;lt;/i&amp;gt;., a Threatened Medicinal Plant

A rapid and improved micropropagation protocol was developed for Curcuma sp., a threatened and high value medicinal plant, using main bud sprout and top of rhizome sprout as explants. Stepwise optimization of different plant is to change the growth regulator, reduce the level of macro-nutrition and add humate. The present study has created multiple shoot and root induction and plantlet in vitro culture, transfers the plantlet to ex vitro. The M2 medium (MS’s macronutrition 1/4, MS’s micronutrition + Morel’s vitamine + coconut milk 10% + sucrose 25 g/l + humate 1.0 ml/l + agar 7.5 g/l + 2,4D 0.5 mg/l + BAP1.0 mg/l + TDZ 1.0 mg/l) is the highest ratio of callus induction. The TA3 medium (MS’s macronutrition 1/4 + MS’s micronutrition + Morel’s vitamin+ coconut milk 10% + sucrose 25g/l + humate 2.0 ml/l + 2,4 D 0.5 mg/l + Kinetin 2.0 mg/l + TDZ 1.0 mg/l + BAP 1.0 mg/l2 + NAA 2.0 mg/l + activated carbon 2.0 g/l) is able to create buds and regeneration multiple bud for Curcuma sp. TA3 medium adding IAA 2 mg/l and IBA 0.5 mg/l has resulted in the highest indices of quantity, healthy shoot and large diameter of roots. A large number of healthy plantlets are induced by the medium of MS’s macronutrients 1/2, MS’s micronutrients full, Morel’s vitamin, humate 3 g/l, coconut milk 150 ml/l, activated carbon 3 g/l, composition phytohormone: IAA 2 mg/l + BAP 2 mg/l + TDZ 0.5 mg/l. Further studies should focus on optimizing the humate concentration for these species of Zingiberaceae. The duration of the research is from 5/2015 to 4/2016 at Faculty of Agriculture and Forestry, Tay Nguyen University, Vietnam.

Effect of MgCl2 and double concentration of Murashige and Skoog medium on in vitro plantlet and root cultures generation in halophytic grasswort Salicornia brachiata

An improved micropropagation protocol has been developed for halophytic grasswort Salicornia brachiata by using double concentration of Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (DMS) supplemented with plant growth regulators. Best shoot bud induction (90 %) from nodal explants was observed on DMS supplemented with 3.0 mg L−1 6-benzyl aminopurine (BAP) and 0.5 mg L−1 Zeatin with >3 shoot buds. Shoot proliferation and elongation was achieved on DMS supplemented with 1.0 mg L−1 thidiazuron (TDZ) and 1.0 mg L−1 α-naphthalene acetic acid (NAA) with multiple shoot buds after 30 days of culture. Best callus response (78 %) was observed when stem explants were cultured on DMS supplemented with 2.0 mg L−1 2, 4-dichlorophenoxyacetic acid and 0.01 mg L−1 BAP. Regeneration from the callus was achieved when callus was cultured on DMS supplemented with 0.5 mg L−1 TDZ. These shoot buds were elongated on shoot proliferation and elongation medium. Elongated shoots (5 cm) could be rooted on DMS supplemented with 0.5–1.5 mg L−1 NAA, 0.5–1.5 mg L−1 indole-3-acetic acid and 0.5–1.5 mg L−1 indole-3-butyric acid. DMS medium supplemented with 0.5 mg L−1 NAA found to be best for rooting and the addition of 20 g L−1 magnesium chloride (MgCl2) to the medium resulted in highest percentage of rooting and not with the addition of sodium chloride (NaCl). Rooted plants could be established in soil with 55 % survival. In another set of study on root culture generation, many roots initiated from explants cultured on DMS supplemented with 2.0 mg L−1 NAA. Addition of 10 g L−1 MgCl2 was highly beneficial in stimulating root initiation and proliferation as compared to NaCl.

Micropropagation and antimicrobial activity of Curcuma aromatica Salisb., a threatened aromatic medicinal plant

A rapid and improved micropropagation protocol was developed for Curcuma aromatica, a threatened aromatic medicinal plant, using rhizome sprout as the explant. Stepwise optimization of different plant growth regulators, carbon sources, and basal media was adopted to establish an efficient micropropagation protocol. When cytokinins, such as benzyl amino purine (BAP) or 6-(a,a-dimethylallylamino)-purine (2iP), were used either singly or in combination with naphthalene acetic acid (NAA) for shoot induction and multiplication, a single use of BAP was the most effective. As a carbon source, 3% (w/v) sucrose exhibits the greatest promotive effect on shoot initiation and proliferation compared with other carbon sources used. Among the basal media, full strength Murashige and Skoog (MS) media produced the best results, compared to other media studied. By using the most effective treatment from each category, an average of 13.2 shoots/per explant were produced after 6 weeks of culture. Moreover, 85% survival was achieved when rooted explants acclimatized ex vitro using a mixture of sterile sand, soil, and farmyard manure (1:1:1). In addition, antimicrobial activities of rhizome extracts were evaluated. Petroleum ether and chloroform extracts of field-grown rhizome showed potential antimicrobial properties against several human pathogenic bacteria including Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Shigella sonnei, and Shigella dysenteriae, with a minimum inhibitory concentration (MIC) ranging from 0.03 to 0.5 mg/mL. Thus, the optimized micropropagation protocol may offer large-scale production of plantlets to meet industrial demand for the rhizome. Moreover, our results suggest the rhizome extract of C. aromatica is a promising antimicrobial agent.

Micropropagation and cytogenetic assessment of Zingiber species of Northeast India.

An improved micropropagation protocol was developed for Zingiber moran and Z. zerumbet, two wild species of the genus Zingiber, found in Northeast India. The effects of growth regulators, sugar concentrations, and nutrients were tested on the rate of shoot initiation and multiplication. An increase in proliferation and multiplication occurred in modified Murashige and Skoog (MS) medium supplemented with benzyladenine and kinetin. About 2 % sucrose and 0.7 % agar were found to be the optimum for shoot multiplication and regeneration. Naphthalene acetic acid at 0.5 mg/L produced the best rooting response for both the species. Regenerated plantlets were acclimatized successfully and cytogenetic stability was confirmed by RAPD profiling and ploidy checks.

Improved micropropagation protocol for leatherleaf fern (Rumohra adiantiformis) using rhizomes as donor explant

A new micropropagation protocol for leatherleaf fern (Rumohra adiantiformis (G. Forst.) Ching) was successfully established using rhizomes as the donor explant, following appropriate sterilization. Murashige and Skoog (MS) medium containing 0.25mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 0.2mg/l α-naphthalene acetic acid (NAA), 1.0mg/l 6-benzyl adenine (BA), and 0.5mg/l thidiazuron (TDZ) with 30g/l sucrose (IM-4) was the most appropriate medium for culture initiation. When entire rhizomes harvested from mother stock plants were cultured on a simple paper bridge containing liquid IM-4 medium, culture initiation improved with 3.4 rhizomes regenerated per sub-culture. The average multiplication rate of newly regenerated rhizomes increased to 5.7/rhizome on half-strength MS medium supplemented with 0.05mg/l IAA, 0.25mg/l BA, 0.5mg/l Kin, 1g/l activated charcoal and 20g/l sucrose (MM-5). The level of multiplication peaked in the fifth subculture and retained high quality until the sixth subculture. From the seventh subculture onwards, the quality of regenerated fronds was reduced. The regenerated rhizomes rooted easily on MM-5 and could be acclimatized ex vitro with 97–100% survival.

Improved micropropagation protocol for maritime pine using zygotic embryos

Pinus pinaster Ait. is a conifer widely distributed through Spain. It is considered one of the major forest species because of wood and resin production. This fact explains the efforts of companies for capturing and enhancing the genetic gain in breeding programs. Micropropagation is a useful strategy to obtain identical plants from selected material, but nowadays, organogenesis protocols are the unique techniques to obtain clonal plants from a high number of elite genotypes. In this work, an improved micropropagation protocol for maritime pine using zygotic embryos as initial explant was developed. To optimize the protocol, the study of culture medium and exogenously applied cytokinins was needed. Embryos cultured on 1/2 DCR as basal medium exhibited high organogenic responses during induction, elongation, and rooting periods. Explants cultured on medium with 2.2 µM meta-topolin showed the highest values in all parameters analyzed along the different micropropagation phases, from induction to acclimatization. These explants showed a survival percentage of 90%, embryos forming shoots of 96.7%, and bud forming capacity and shoot elongation capacity of 4.38 and 3.39, respectively. Moreover, 70% of rooted shoots were obtained. All rooted plants survived in the acclimatizing stage after two months under ex vitro conditions. Use of 1/2 DCR as basal medium may provide an efficient and simplified in vitro protocol from whole zygotic embryos, ensuring the large-scale production of Pinus pinaster superior genotypes.

Multiple adventitious shoot formation in Spanish Red Cedar (Cedrela odorata L.) cultured in vitro using juvenile and mature tissues: an improved micropropagation protocol for a highly valuable tropical tree species

Cedrela odorata L. is a valuable tropical tree widely appreciated for its wood. This species confronts serious problems due to both overexploitation of its natural populations and its susceptibility to the Meliaceae borer Hypsipyla grandella, which destroys the apical meristems and produces structural deformations. The rapid introduction of new varieties through clonal forestry has been demonstrated to be the most effective way to improve the production of perennial plantation species. In this work, we report both a protocol for the rejuvenation of elite mature trees of C. odorata and the optimization of an in vitro culture system to scale up micropropagation. Several media formulations and the use of temporary immersion culture in bioreactors were evaluated. The addition of 20% coconut water to TY17 medium increased the number of adventitious shoots from hypocotyl segments to an average number of 4.68 shoots per explant. To replace coconut water and to define the culture medium, several cytokinins were tested at various concentrations; however, none of them produced the effect of coconut water. Rejuvenation of elite mature individuals was investigated by ex vitro grafting of mature tree twigs onto 3-mo-old juvenile trees. Although the grafting had a positive effect on the micropropagation of mature material, the multiplication rate of 1.5 new shoots per explant did not compare to the organogenic capacity of younger materials. Shoot and root elongation as well as acclimatization to ex vitro conditions were carried out in a temporary immersion culture of juvenile material using BioMINT® bioreactors. A 3.5-fold increase in shoot elongation and a 4-fold increase in root elongation were achieved compared to material cultured on semisolid media. Furthermore, this culture system allowed for 98% effectiveness in the soil adaptation of the in vitro-grown plants. The scaled-up multiplication capacity over a period of 6 mo calculated for the system is above 16,000 plants per mother plant with young materials but is only 125 with mature materials.

An improved micropropagation protocol for maritime pine (Pinus pinaster Ait.) isolated cotyledons

Summary In this work an improved micropropagation protocol for Pinus pinaster Ait. using isolated cotyledons is presented. The variables studied during bud induction were embryo preculture, type and concentration of cytokinin and infl uence of illumination culture conditions. Shoot development and proliferation was obtained by transfer to hormone-free medium with activated charcoal. This protocol also describes a procedure for successful continuous multiplication using axillary shoots and brachyblasts formed during shoot development. Based on 86 per cent rooting success rate, at least 70 plantlets can be expected per seed after 16 weeks.