Efficient Micropropagation Method Research Articles

Efficient In Vitro Platform for Multiplication, Acclimatization, and Deliver of High-NaCl-Tolerant Clones of the Halophyte Arthrocaulon macrostachyum

Halophytes are species able to grow and complete their life cycle under NaCl concentrations above 0.2 M. In a context of growing population and uncertain agricultural food sustainability, there is an interest on halophytes as potential source of food and fodder. However, scarce research has addressed in vitro propagation of halophytes as a tool for study, conservation, and propagation of elite germplasm. In this study, an efficient method for micropropagation of the succulent and salt accumulator halophyte Arthrocaulon macrostachyum has been established for the first time, using shoot tips from in vitro-germinated plant material. During shoot multiplication stage, superior genotypes were selected from explants grown in high strength and NaCl content medium and subsequently rooted and acclimatized to ex vitro conditions. A comprehensive characterization including determination of oxidative stress, photosynthesis performance, and mineral nutrient contents was done. This research gains insight into the physiological and biochemical characterization of halophytes during micropropagation and provides a solid platform for the production of elite A. macrostachyum germplasm for ulterior uses.

An Efficient Method of Pennisetum × advena ‘Rubrum’ Plantlets Production Using the Temporary Immersion Bioreactor Systems and Agar Cultures

The aim of this study is to develop an efficient method for micropropagation of Pennisetum × advena 'Rubrum'. Agar cultures containing Murashige and Skoog (MS) medium supplemented with 6-benzyl-amino-purine (BAP) in various concentrations (0.5 mg/L to 2 mg/L) and a temporary immersion bioreactor system (TIS) using liquid medium MS with an addition of 1 mg/L BAP were tested. Rooting was performed using ½ MS medium supplemented with different auxin combinations (indole-3-butyric acid IBA and α-naphthalene acetic acid NAA) and activated charcoal. The TIS method was found to be the most efficient, producing 36.9 new plants within four weeks. The resulting plantlets were thin and bright green in color, with no signs of hyperhydricity. The most suitable agar medium yielded 19.5 new plants within eight weeks. For rooting, ½ MS supplemented with 0.5 mg/L IBA and 0.5 mg/L NAA exhibited an 84% rooting rate, whereas the addition of activated charcoal inhibited rooting.

An efficient indirect plant regeneration from shoot apical meristem (SAM) derived embryogenic callus of Miscanthus × giganteus

The perennial grass Miscanthus × giganteus with its high biomass and biofuel yields on marginal lands is a leading feedstock contender for future low carbon emission bioenergy production. Most M. x giganteus grown is from a sterile clone of a triploid form. Although this has the advantage that its production is not invasive, most plantings have had to be via rhizomes, which is time-consuming and expensive. Here we report an efficient micropropagation method via indirect plant regeneration from embryogenic callus developed from shoot apical meristem (SAM) explants of two field-grown elite M. × giganteus genotypes, Illinois and Ogi80. Following an optimized surface disinfection of field-grown stem, avoiding subsequent necrosis of explants, non-contaminated SAM in vitro culture was established. We successfully demonstrated in vitro differentiation of SAMs into somatic embryos on SCIM-22 medium, containing p-chlorophenoxyacetic acid and α-naphthalene acetic acid as growth regulators, which activity was synergistically amplified with activated charcoal, putrescine and 2-aminoindane-2-phosphonic acid. We observed 49% (Ogi80) and 82% (Illinois) embryogenic calli from the calli initiated, which developed into plantlets with 12% (Ogi80) and 33% (Illinois) of total regeneration efficiency after 15 weeks of culture. In vitro regenerated plants grown till maturity showed biometric traits comparable to rhizome-propagated plants, but in vitro regenerated plants of genotype Illinois were significantly superior regarding stem diameter, leaf width and tillering. This study is the first demonstration of somatic embryogenesis from vegetative tissues of field-harvested M. × giganteus resulting in propagation competitive with rhizome-propagated plants in terms of yield potential.

In Vitro Propagation of the Mount Parnitha Endangered Species Sideritis raeseri subsp. Attica

Over the past few decades, both wildfires and human-sparked fires have ravaged Mount Parnitha, destroying the mountain’s unique natural ecosystem, applying pressure to its flora, and subjecting the vulnerable populations of Sideritis raeseri subsp. attica to excessive stress. The present study aims to establish an efficient micropropagation method starting from in vitro-grown seedlings. The in vitro germination study carried out during the production of seedlings revealed a higher germination rate (34.0% and 37.0%, respectively) at 20.0 °C and 25.0 °C. The in vitro-derived seedlings studied were used as the starting material for the establishment of various media. Murashige and Skoog (MS) media, hormone-free and containing 0.5 mg L−1 6-benzyladenine (BA), led to the satisfactory (84.0–89.0%) establishment of plantlets. During the multiplication phase, the study used BA in conjunction with α-naphthaleneacetic acetic acid and four different cytocinins (BA; kinetin (KIN); 6-(γ-γ-dimethylallylamino) purine; zeatin) at low concentrations (0.5 mg L−1). During the second subculture, a high multiplication index (7.3 and 6.4, respectively) was found for the hormone-free MS medium and the MS medium containing KIN at 0.5 mg L−1. Hyperhydricity took place on the media supplemented with hormones. Rooting occurred on the half-strength MS medium (51.0%). After two months, the plants’ survival rate stood at 100.0%, as did their ex vitro acclimatisation rate, which also registered at 100.0%. The present results could encourage not only the introduction of S. raeseri subsp. attica into the industry of floriculture as a new ornamental plant but also its ex vitro conservation.

Re-introduction of an extinct population of Pulsatilla patens using different propagation techniques

The study focuses on the propagation of a rare and endangered plant species (Pulsatilla patens) to re-introduce an extinct population from calamine area in Southern Poland. The plants were propagated from seeds, rhizome cuttings, or regenerated in vitro from shoot tips, hypocotyls with roots or cotyledons of seedlings on Murashige & Skoog (MS) medium supplemented with 0.25 or 0.50 mg L−1 BAP (Benzylaminopurine) via direct and indirect organogenesis or somatic embryogenesis (SE). The most efficient micropropagation method was with shoot tips as an explant on MS + 0.25 mg L−1 BAP where 97% of the explants produced multiple shoots, mass SE was observed after transfer on ½ MS with 2% saccharose; 267 (35%) shoots rooted on ½ MS + 2% saccharose were acclimatized to ex vitro conditions. Flow cytometry revealed genome size stability of propagated plantlets. Low genetic differentiation between micropropagated plantlets and initial material was indicated by ISSR (Inter Simple Sequence Repeat) markers. Totally, 132 vigorous plantlets obtained on various pathways were introduced to the field plots in 2020; 30.33% survived the winter, and several reached the generative stage and flowered in the spring 2021. In next season (March/April 2022) the number of introduced plants decreased to 25% while the number of flowering and fruiting shoots in different clumps increased in some plots. This is the first report of successful re-introduction of the endangered P. patens based on micropropagation, rhizome cuttings, and seed germination.

Effect of vermicompost extract with phytohormones on micropropagation and in vitro flower induction of Lindernia minima (Benth.) Mukerjee - an endangered plant species endemic to Tamil Nadu, India

Lindernia minima is an endangered and endemic plant species (Red list data of IUCN). Based on the IUCN threat status and highly restricted distribution, the present research work develops an efficient micropropagation method for its conservation. The present study employed purified vermicompost extract in combination with different concentrations of phytohormones on plant regeneration and in vitro flower induction. The shoot tip explants were cultured on quarter-strength of MS medium (Murashige and Skoog) with different (0.5 – 3.0 mg L−1) concentrations of BA (6-Benzyladenine), KIN (Kinetin), and TDZ (Thidiazuron). Initially, the effect of phytohormones alone produced the lowest results with reference to the percentage of response, number of shoots/explant, and length of the shoots. Hence, the explants were inoculated on a quarter-strength MS medium with various concentrations of phytohormones combined with various percentages (25%, 50%, and 75%) of purified vermicompost extract (VCE). TDZ at 2.5mg L−1 with 50% of VCE produced a high-frequency rate of the shoots. Elongated shoots were transferred into a quarter-strength of MS medium with TDZ (2.5 mg L−1) and 50% of VCE, and fortified with several concentrations of Gibberellins (0.2 – 1mg L−1) for in vitro flower induction. After 4 weeks of culture, the maximum numbers of shoots with well-developed flowers were observed at 2.5 mg L−1 TDZ fortified with 50% VCE and 0.6 mg L−1 of GA3. Well-elongated in vitro shoots were inoculated onto MS rooting medium with various concentrations of (0.5 – 3 mg L−1) IAA (Indole -3- acetic acid) and IBA (Indole -3- butyric acid). The highest root formation was observed at 2 mg L−1 IBA with a quarter-strength MS medium. After the well-root formation, plants were transferred to the acclimatization process and established with a 95% of survival frequency in the field conditions. The present research revealed that adding purified VCE with phytohormones promoted the high regeneration ability of Lindernia minima from shoot tip explants. So far, in vitro propagation studies are not reported for this plant. Hence, the present investigation will be more useful for mass in vitro clonal propagation of Lindernia minima using shoot tip explants.

The effect of 2,4-dichlorophenoxyacetic acid, benzyl amino purin and cupric sulphate on in vitro propagation system from shoot apices of shoot tiller of hybrid Napier grass (Pennisetum purpureum Schum)

An efficient micropropagation method of hybrid Napier grass (Pennisetum purpureumn Schum) for in vitro plant production and material breeding was established from multiple-shoot clumps (MCS) regeneration system. This system was important for forage breeding system. Shoot apices from shoot-tillers produced MSC on Murashige-Skoog (MS) induction medium containing several combinations of BAP and 2,4-D in induction stage. The addition of 5 μM (v/v) and 50 μM (v/v) CuSO4 were added in best medium for inoculation to proliferate the clump in proliferation/multiplication stage. Plant regeneration was achieved by culturing on solid MS with several combination of medium containing NAA and BAP in regeneration stage. The best results for induction were Murashige-Skoog (MS) induction medium containing 2 mgL−1 BAP and 0.1 mgL−1 2,4-D. The proliferation stage on MS medium containing 5 μM CuSÜ4 effective for proliferation (50% multiple shoot formation). The regeneration stage using 0.1 mgL−1 NAA and 2.0 mgL−1 BAP (51.6% number of shoot can regenerate). All plantlets were successfully grown up in an acclimatization stage. Based on the results, the hybrid Napier grass regeneration via MSC was a stable tissue culture system (no albino plats), which could be applied either for further genetic transformation assay or for alternative supply of nursery plant in the future.

Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

A novel temporary immersion bioreactor system for large scale multiplication of banana (Rasthali AAB\u2014Silk)

Musa sp. cultivar Rasthali (Silk AAB) is a choice variety of the Asian sub-continent. Its production and sustenance are threatened by Fusarium wilt, which affects the livelihoods of small and marginal farmers. The use of quality planting material is one of the strategies to manage the disease. Availability of quality planting material for varieties other than Grand Naine is limited. Large-scale micropropagation using existing technologies is laborious and expensive. Temporary immersion bioreactor system is emerging as a potential advancement in the micropropagation industry. In this study, a cost-effective temporary immersion bioreactor (TIB) system has been developed and an efficient micropropagation method has been standardized. Explants cultured in TIB with 250 ml of culture medium in a 2-min immersion frequency of 6 h were found to be efficient for shoot proliferation and rooting. Its efficacy has been compared with the semisolid culture method. At the end of the 6th subculture, 1496 ± 110 shoots per explant were obtained in TIB. Chlorophyll, carotenoid, stomatal index, and the number of closed stomata were examined to determine the physiological functions of the plants grown in TIB and compared with semisolid grown plantlets. Plantlets grown in TIB were genetically stable and were confirmed using inter-simple sequence repeat (ISSR) markers. The multiplication of shoots in TIB was 2.7-fold higher than the semisolid culture method, which is suitable for large-scale production of planting material for commercial applications.

Callus Induction and Micropropagation of Lilium candidum L. Using Stem Bulbils and Confirmation of Genetic Stability via SSR-PCR

Natural populations of Lilium candidum L. are remarkably affected by biotic and abiotic factors therefore there is a requirement to develop effective micropropagation protocol to provide mass production, multiplication and conservation of these plants. For this reason, this study was aimed to develop an efficient micropropagation method for multiple shoot production via somatic embryogenesis induced from L. candidum stem bulbils and also to determine the genetic stability of in vitro grown plants using SSR markers. The obtained results of this study are the first comprehensive reports including an investigation of genetic fidelity on somatic embryogenesis of L. candidum. After surface sterilization of bulbils, the calculated regeneration percentage of them was 89.5% and the callus induction was achieved using leaf segments of in vitro grown bulbils. The well formed somatic embryos were obtained from smooth whitish-yellow colored calli and these somatic embryos produced well formed healthy L. candidum seedlings with adventitious roots. All rooted seedlings were easily adapted to greenhouse conditions and the genetic stability of in vitro grown seedlings were determined by using SSR-PCR technique and it was calculated as 100%.

Efficient Micropropagation of Chestnut Hybrids (Castanea spp.) Using Modified Woody Plant Medium and Zeatin Riboside

The chestnuts genus (Castanea spp.) is comprised of economically important trees native to the Northern hemisphere that are used as food and hardwood timber. Here, a very efficient method for micropropagation of European × Japanese chestnut hybrids (Castanea sativa × C. crenata) is described. Woody Plant Medium was used as the basal medium. In vitro shoots of four rootstock cultivars were micropropagated without shoot-tip necrosis on multiplication medium containing 5.7 or 11.4 µmol·L−1 zeatin riboside, and were rooted on rooting medium containing 2.46 µmol·L−1 indolebutyric acid. Monthly shoot multiplication rates for each cultivar were 2–5 folds. In vitro rooting percentages for four cultivars were 87% for ‘Maraval’, 67% for ‘Marigoule’, 93% for ‘Marsol’, and 97% for ‘Précoce Migoule’. Within a 5 week period, 80%–95% of rooted shoots were successfully acclimated under high humidity conditions after they were planted in either soil or rockwool.

In vitro propagation of Farsetia macrantha Blatt. & Hallb.: an endemic and threatened plant of Indian Thar Desert

Farsetia macrantha Blatt. & Hallb. is an endemic and critically endangered plant species of Indian Thar desert. Being an endemic and threatened plant species, there is need to develop an efficient in vitro regeneration protocol for its conservation. Since, there is no published report on in vitro regeneration of F. macrantha, therefore, present study was designed to develop an efficient micropropagation method of F. macrantha along with foliar micromorphological studies. Cotyledonary node taken from 15 to 20 days old seedlings was used as an explant for in vitro regeneration. MS medium supplemented with additives ascorbic acid (50 mg l−1), citric acid (25 mg l−1), l-arginine (25 mg l−1) and adenine sulphate (25 mg l−1) and 0.5 mg l−1 BA was best for shoot bud induction. Different types, concentrations and combinations of cytokinins (BA, Kin and mT) and auxin (IAA) tested for shoot multiplication, maximum number of shoots (25.10 ± 0.69) regenerated on MS medium containing additives and combination of two cytokinins 0.5 mg l−1 BA + 0.25 mg l−1 Kin. Microshoots were rooted successfully with highest rooting response (75.59%) on ½ MS medium containing 2.0 mg l−1 IBA. In vitro rooted plants were hardened with ~ 75% survival rate. Comparative foliar micromorphological studies conducted among in vitro grown and hardened plants suggest the developmental changes of micropropagated plants during acclimatization. The present in vitro regeneration protocol could be used for large-scale propagation and conservation of F. macrantha. Farsetia macrantha is an endemic and critically endangered plant species of Indian Thar desert and presently it is restricted only to the Barmer district of Rajasthan, India. In the present study, an in vitro regeneration method has been developed first time for its conservation.

PLANT TISSUE CULTURE IN MUNTINGIA CALABURA IN VITRO CLONAL PROPAGATION, CALLUS INDUCTION AND GERMPLASM CONSERVATION OF ‘CERECILLO’ MUNTINGIA CALABURA L. (MUNTINGIACEAE)

Muntingia calabura L. (Muntingiaceae) is a fast-growing tree native to tropical America, abundant in the seasonally dry forest of the north coast of Peru. Tissue culture is an effective procedure to produce healthy plants, rapid clonal propagation and several morphogenic process. The objective of this study was to formulate an efficient method for micropropagation, morphogenesis callus induction and germplasm conservation of this species. In this study, seeds, shoot-tips and nodal segments of seedlings were used as explants, inducing various morphogenic processes in different combinations of growth regulators and osmoregulatory substances. In vitro seeds germination was 100% up to four months after the ripe fruits were collected and after 12 months the germination rate was 0.0%. The highest elongation of shoot was observed with 0.5 mg L-1 2iP (3.11 cm) although the highest number of shoots formed (18.0 and 16.5) was observed with 0.5 mg/L KIN or TDZ, respectively, after 30 days of culture. The best callus induction was obtained in 0.5 and 1.0 mg L-1 TDZ, 1.0 and 2.0 mg L-1 2,4-D, 2.0 mg L-1 NAA or 2.0 mg L-1 NAA with 0.1 and 0.5 mg L-1 BAP, after 45 days of culture period. Shoot regeneration (> 10 shoots/explant) was observed with 0.1 to 2.0 mg L-1 NAA. Root induction was observed in all shoots cultured in various concentrations of IBA and NAA-GA3, after 30 days of culture. After two months, well rooted plantlets were transplanted in greenhouse conditions, however, the survival rate was less than 10%. Only in treatment with mannitol 2.0% in explants without roots, the highest in vitro conservation rate (50%) was reached, after 6 months of culture, while in the control treatment in culture medium without ABA and mannitol, but supplemented with 0.02-0.02 mg L-1 (IAA-GA3), the conservation rate reached 100%. The results demonstrated the applicability of tissue culture in the micropropagation and in vitro germplasm conservation of M. calabura.

Callus induction and high frequency organogenesis in saffron (Crocus sativus L.)

Saffron (Crocus sativus L.) is an economically important plant with a very slow natural multiplication rate and plant tissue culture is expected to enhance its reproductive capacity. In present study, corm explants were used for micro-propagation. Auxins and cytokinin were found important for in vitro propagation of explants. Overall, 93% calli were induced from meristematic region of corm explants propagated on Murashige and Skoog (MS) medium supplemented with 2 mg L−1 2,4-dichlorophenoxy-acetic acid (2,4-D), 0.5 mg L−1 kinetin (Kn), 2% sucrose and 100 mg L−1 ascorbic acid after 6 weeks inoculation. Somatic embryogenesis was observed on half strength MS medium supplemented with 2 mg L−1 indole-3-acetic acid, 2 mg L−1 Kn and 100 mg L−1 ascorbic acid. Organogenesis from calli was observed with 89.6% shooting on MS medium containing 2 mg L−1 Kn and 2 mg L−1 IAA. Adventitious rooting of 91.6% calli was obtained on MS medium supplemented with 2 mg L−1 indole-3-butyric acid. There are plenty of protocols available for tissue culture of saffron but this protocol provides an easy and time efficient method for micropropagation of saffron in vitro.

In vitro tissue culture and genetic analysis of two high-CBD medical cannabis (Cannabis sativa L.) breeding lines

The species Cannabis sativa L. has recently witnessed a resurgence of interest all over the world due to its multipurpose applications and the scientific confirmation of its pharmacological properties. Genotypes with high cannabinoid content are appreciated in the pharmaceutical and cosmetic industries due to their therapeutic potential. These genotypes, with predominantly high cannabidiol (CBD) content, are the subject of research and breeding in several programs, but to date, little data is published on the in vitro tissue culture of cannabis. Our study focused on the establishment of an efficient micropropagation method for two high-CBD breeding lines (MX-CBD-11 and MX-CBD-707) as the basis for advanced biotechnological breeding approaches. The results demonstrated that the in vitro culture of medical cannabis can be initiated on different culture media, that cultured plants can be successfully acclimatized, and that nodal position, and especially the genotype, have a significant influence on the success of shoot culture establishment. They showed that the published tissue culture media optimized for one high-THC strain of Mexican cannabis are not as efficient for other genotypes of (medical) cannabis. We complemented this research with a genetic study of 95 plants of the two breeding lines with 16 microsatellite (SSR) markers which clustered the plants based on breeding line. The results demonstrated that only 8 markers are needed for discrimination of all analyzed plants and their usefulness for clonal identification.

In Vitro Regeneration of Coral Tree from Three Different Explants Using Thidiazuron

The coral tree (Erythrina variegata) is a multipurpose horticultural plant with a plethora of medicinally important alkaloids. Regeneration via tissue culture can provide an efficient alternative to seed-grown plantlets and reduce the cost of the plant significantly. Thidiazuron (TDZ) is an efficient plant growth regulator and is effective in numerous species. However, the response to it varies with the type and position of the tissue on the plantlet treated. This study was carried out to ascertain the best tissue types for micropropagation of the coral tree using TDZ. Three tissue types (shoot tip, nodal, and hypocotyl), originating from different strata of the plantlet were evaluated. Adventitious shoots were observed in all three explants at the tested concentrations. However the quality and the shoot number varied significantly with the type of explant. Explants with a meristematic zone (shoot tip and nodal) were more responsive to the treatment compared with hypocotyl tissue lacking preexisting meristem. Nodal explants produced the maximum number of shoots (about eight) per explant after 4 weeks of culture, whereas shoot tips produced about only five shoots per explant at an equimolar concentration (1.5 µm). Approximately three shoots were observed in hypocotyl explants. Moreover, growth and rooting of the regenerated shoots was influenced by the origin of the explants. The molecular characterization of the regenerants using intersimple sequence repeat (ISSR) markers revealed genetic homogeneity among regenerants. An efficient micropropagation method for the coral tree is described.

An improved micropropagation system, ex vitro rooting and validation of genetic homogeneity in wild female Momordica dioica: an underutilized nutraceutical vegetable crop.

Momordica dioica Roxb. ex Willd., isa perennial and dioecious (2n=28) plant of family Cucurbitaceae. Conventional methods of propagation through seeds, stem cuttings and rhizomatous/tuberous roots are inadequate for its mass cultivation as a vegetable crop. This paper reports an improved and efficient micropropagation method for wild female M. dioica using nodal explants. Shoot amplification was achieved using subculturing of in vitro raised shoots on MS medium supplemented with various concentrations of 6-benzylaminopurine (BAP) alone or in combination with indole-3-acetic acid (IAA). The maximum number of shoots (45.30±3.83) with an average length 6.52±0.89cm were differentiated on MS medium containing 0.5mgL-1 BAP, 0.1mgL-1 IAA and additives (50mgL-1 ascorbic acid, 25mgL-1 each of adenine sulphate, citric acid and l-arginine). The cloned shoots were rooted ex vitro. Each shoot treated with 250mgL-1 IBA for 5min produced 12.3± 1.33 with a mean length 5.4± 0.73cm. More than 85% (46 plants) of ex vitro rooted plantlets were successfully hardened in a greenhouse with normal growth characteristics. In order to evaluate the genetic stability of micropropagated plants, the two PCR-based techniques, Random Amplified Polymorphic DNA(RAPD) and Inter Simple Sequence Repeats(ISSR) were used. The amplification patterns of the micropropagated and mother plant were monomorphic thus depicting genetic stability of the micropropagation system. This protocol could be effectively employed for the mass multiplication of wild female M. dioica, a popular summer vegetable crop.

Micropropagation and Assessment of Genetic Stability of In Vitro Raised Jojoba (Simmondsia chinensis Link.) Plants Using SCoT and ISSR Markers

This study is aimed at developing an efficient method for micropropagation of true-to-type jojoba plants. Nodal segments were used for in vitro shoots proliferation. Among three concentrations of BA used for multiplication, 1 mg/l BA gave the highest number of shoots. To enhance growth of shoots, combinations of BA and Kn were investigated. The greater value of shoot length and the maximum number of nodes were observed in the medium containing 1 mg/l BA + 1.5 mg/l Kn. Among different medium used to increase the rate of multiplication, the maximum number of shoots was recorded at ½ MS + Gamborg B5 (B5) vitamins + 2 mg/l Kn + 10 mg/l adenine sulphate (AS). Rooting was obtained upon supplementation of ½ Woody Plant Medium (WPM) with 1 mg/l IBA. Acclimation was achieved by transplanting rooted plantlets into pots containing peat moss and vermiculite. Start codon targeted (SCoT) and intersimple sequence repeat (ISSR) analyses were carried out to assess the genetic fidelity of micropropagated plantlets. All banding profiles of the two analyses from micropropagated plants were monomorphic and similar to the mother plant. Hence, this protocol can be employed for commercial micropropagation of jojoba without the risk of losing genetic stability.Plant Tissue Cult. & Biotech. 25(2): 165-179, 2015 (December)

Micropropagation of wild species of the genus Asparagus L. and their interspecific hybrids with cultivated A. officinalis L., and verification of genetic stability using EST-SSRs

An efficient micropropagation method for asparagus species was developed in this study. The method allows the fast cloning of the elite genotypes from different asparagus species and the interspecific hybrids obtained from these species. Rhizome bud explants were disinfected using 3 g l−1 of benomyl and 20 g l−1 of sodium hypochlorite. Then, they were cultured on Asparagus Rhizome Bud Medium 3 (ARBM-3) consisting in modified Murashige and Skoog medium with salts with EDDHA-Fe (85.7 mg l−1) instead of EDTA-Fe and vitamins, supplemented with 0.3 mg l−1 NAA, 0.1 mg l−1 KIN, 2 mg l−1 ancymidol and 6 % sucrose. Results showed that the method developed produced high disinfection rates (70–95 %). More than 70 % of the explants developed shoots and the rooting rate on ARBM-3 medium was 30–45 %. The rooting rate increased to 60–85 % when the unrooted shoots were subjected to an additional cycle of rooting, reaching 100 % after two cycles of rooting. The multiplication was achieved through mechanical division of rooted shoot clusters growing in ARBM-3. The acclimatization rate of the micropropagated plantlets was higher than 90 %. The micropopagated plantlets were screened for somaclonal variation using 12 expressed sequence tag derived simple sequence repeat markers. The results confirmed the character “true to type” of the plantlets, indicating that the method developed in this study can be used to successfully micropropagate asparagus species.

MICROPROPAGATION OF AMERICAN ELDERBERRY: DISINFECTION OF EXPLANTS

The fruits and flowers of the American elderberry (Sambucus Canadensis) hold potential as a source of food and nutraceutical products. Consequently, commercial scale orchards are beginning to appear in North America. Research to produce new cultivars and identify promising specimens from wild populations is currently underway in response to an increasing demand for large scale production of elite plants. Using only a limited number of mother plants, an efficient micropropagation method could satisfy such a demand. A detailed procedure for micropropagation of American elderberry is presented. In this first part, various disinfection procedures were tested to control the high internal contamination level observed with elderberry explants. Whole nodal sections containing two buds proved to be the most successful material giving a fast growth and a satisfactory multiplication rate. The best results were obtained when nodal sections were disinfected in a 3% solution of sodium hypochlorite then soaked in a 0.5% silver nitrate solution followed by a final bath in a solution of citric and ascorbic acid. The need for a strong disinfection procedure resulted in a low survival rate with some cultivars. The proposed disinfection procedure was judged satisfactory for commercial purposes. Medium optimization is presented in a second paper.