Shoot Multiplication Rate Research Articles

Silicon nanoparticle(s) induced morpho-anatomical traits and improved micropropagation of white fleshed dragon fruit [Selenicereus undatus (Haworth)]

Silicon nanoparticles (SiNPs) improve plant growth and yield by enhancing nutrient uptake and stress tolerance. These nanoparticles boost plant defense mechanisms against pathogens and environmental challenges. In this study, seedlings-derived explants of white-fleshed dragon fruit (Selenicereus undatus) were used for differentiation and high-frequency shoot regeneration on Murashige and Skoog (MS) medium with 6-benzylaminopurine (BAP, 0.5 mg L−1) and α-naphthalene acetic acid (NAA, 0.25 mg L−1). The complementation of 3.0 mg L−1 SiNPs in the optimized nutrient medium enhanced the shoot multiplication rate to 23.0 cladodes per explants. The regenerated cladodes were structurally well-suited with thick cuticles, well-developed epidermis, hypodermis, aquiferous cortex, and collateral vascular bundles compared to the control and other concentrations of SiNPs tested. SiNPs promoted the development of 3-5 aerial and adventitious roots per cladode enabling preferred direct hardening of cloned plants on soilrite ex vitro. The morphological transformations improved by SiNPs, including the formation of robust cladode and spines, played a pivotal role in achieving a high rate of acclimatization success (96%) in the field. The incorporation of SiNPs in vitro at the shoot amplification stage of micropropagation of S. undatus induced morphogenesis and structural changes, and promoted qualitative and quantitative improvements in the regenerated cladodes which helped in enhanced survival of plants in vivo.

In vitro morphogenesis and micro-morpho-anatomical developments in Moringa concanensis Nimmo.: An endemic tree of Indian sub-continent

Moringa concanensis Nimmo. is a regionally important and underutilized medicinal tree of the Indian subcontinent. An in vitro morphogenesis protocol for Moringa concanensis has been developed using seedling-derived explants. The cotyledonary node explants were found to be the most suitable exhibiting cent-percent bud break with each regenerating 2.33±0.76 shoots of 1.32±0.69 cm length within 15 days on 1.0 mg/L 6-benzyl aminopurine (BAP) supplemented modified Murashige and Skoog Medium (MMS) media. The rate of shoot multiplication increased up to 10.17±2.54 shoots/inoculum by sub-culturing of regenerated shoots on 0.5 mg/L BAP along with 0.1 mg/L Indole acetic acid (IAA). The in vitro cloned shoots were rooted using both in vitro and ex-vitro methods by applications of root-inducing auxins, indole butyric acid (IBA), and naphthalene acetic acid (NAA). IBA proved to be more effective and induced roots in all in vitro raised shoots. It produced 17.72±4.83 roots/shoot with 2.12±0.88 cm length on 1/4th strength MMS media fortified with 1.0 mg/L of IBA. Ex vitro rooted plantlets with concurrent acclimatization were achieved by pre-treating shoot bases with IBA at 250 ppm for 5 min. Comparative micro-morpho-anatomical evaluation of leaf and stem revealed differentiation of non-glandular trichomes, anomocytic stomata, and vascular tissue development under applied in vitro conditions, which suggests the survivability of plantlets. Sixty percent of the in vitro regenerated plants survived in the field. It is the foremost report on in vitro morphogenesis of Moringa concanensis protocol using cotyledonary nodal explant and ex vitro rooting method. The tissue culture methods defined and developed can be used for large-scale multiplications of Moringa concanensis as non-conventional approaches.

In Vitro Regeneration, Conservation, and Field Evaluation of a Medicinal Plant– Greater Burdock (Arctium Lappa L.)

A suitable micropropagation protocol and ex vitro acclimation method have been developed from in vitro grown seedling explants through cotyledonary node and leaf explants in consideration of the vegetable and medicinal properties of Greater Burdock. MS medium with 0.5-4.0 μM BAP showed highest percentage of axillary shoot regeneration from the cotyledonary nodal explants. Direct shoot regeneration was achieved by culturing 1.0 cm2 sections of about 25 days old leaves of in vitro grown shoot on MS medium enriched with 4.0 μM BAP and 2.0 μM IBA or NAA after 5 weeks of culture. Within six weeks of incubation on medium enriched with 4.0 M BAP and 2.0 M IBA or NAA, the leaf explants also developed callus from the cut margins. The greatest number of adventitious shoots could then be formed from the leaf-derived callus within 10 weeks of culture on the same media mix. More than 20 shoots were formed per callus clump at the third subculture, which had the highest rate of shoot multiplication. A. lappa's shoot and callus were both preserved at 5 ºC in MS medium with 4.0 μM Kn and 2.0 μM IBA, as well as 4.0 μM BAP and 2.0 μM IBA, respectively. The in vitro proliferated and elongated shoots were separated from callus clump for rooting. A root-induction MS medium with 6.0 μM IBA or NAA was used to cultivate the microshoots individually. All of the cultured microshoots generated 2-16 roots within 4 weeks of being moved to the rooting medium. Regenerated plantlets were transferred to vermiculite and successfully established in an ex vivo environment with a 98% survival rate. J. Bio-Sci. 31(1): 1-15, 2023

In vitro multiplication of Pothos tener shoots

South Sulawesi-endemic Pothos tener can be used as a raw material for medicine because it contains antimicrobial compounds. To date, plants have been collected directly from the wild, without any cultivation or propagation measures. This will threaten its continued existence. Micropropagation is an efficient method for propagating plants. Multiplication of shoots is an essential step in micropropagation. Explants can be cultured on media containing cytokinins to increase the number of shoots that serve as new plant material. This study investigated the effect of cytokinins on the in vitro multiplication of Pothos tener shoots. The growth medium utilized was MS media (1962), which was added with BAP, Kinetin, 2iP at concentrations ranging from 0 to 2.5 mg/l. Every treatment was administered 10 times. This research indicates that the rate of shoot multiplication increases as cytokinin concentrations rise. However, the best results were obtained on MS media added with 2 mg/l BA, as shoot height, number of shoots, and number of leaves were all increased. At a concentration of 2.5 mg/l, fewer shoots were produced, and their growth was stunted.

Liquid Culture System: An Efficient Approach for Sustainable Micropropagation

Micropropagation of important forestry, horticultural and medicinal plants have made revolutionary changes in terms of research and commercialization. However, there are a variety of factors which influence the scaling-up and commercialization aspects, which decide whether mass propagation will be effective and lucrative. Low rates of shoot multiplication, increased costs of media components, loss of cultures due to contamination, and difficulties with hardening and acclimation are the key obstacles to scaling up micropropagation technology. These restrictions have forced a large number of in vitro technologies developed for a range of plant species to be used only under research laboratories settings. To apply tissue culture technology to large-scale propagation, it is required to develop techniques that are relatively simple to adopt, have high multiplication rate with high levels of reproducibility, and exhibit higher survival of plantlets when transferred to ex vitro conditions. Efficient techniques include utilization of liquid culture systems and replacement of agar with other gelling agents. These techniques allow development of micropropagules that not only function better in post-vitro soil conditions and are comparatively less expensive, but will also help develop a workable micropropagation technique that can be applied to the mass production of desirable plant species. The current review describes liquid culture system as an efficient approach to produce large number of plants at low production cost.

Revisiting In vitro Micropropagation Protocols of Mimosa pudica for Enhanced Seed Germination, Shoot Multiplication, and Root Initiation

Mimosa pudica is a medicinal plant worthy of therapeutic properties. It is often overlooked as one of the weed species, and the potential was underappreciated despite its abundance in nature, particularly in tropical climate countries. Considering the aptitude of this species, the micropropagation protocol of M. pudica was revisited and enhanced. The seed surface sterilization and germination were assessed, followed by shoot multiplication rate and root initiation efficiency. Seeds of M. pudica were best surface sterilized with 35% of Clorox and recorded the highest germination rate at 65.55% in media of three-quarter strength Murashige and Skoog (MS) combined with 30 g of sucrose. The explants were harvested and subcultured into multiplication media fortified with 6-benzylaminopurine, kinetin, and coconut water. The highest number of new shoots and leaves were obtained at 3 shoots and 3.27 cm in media of MS combined with 17.6 μm kinetin. Half-strength MS media devoid of hormone was recommended for root induction, resulting in the 5 healthy roots at 2.06 cm after 4 weeks of culture. Overall, the developed micropropagation protocol of M. pudica could facilitate its large-scale cultivation, indicating its potential as a medicinal crop for the extraction of bioactive compounds.

A Comparison of Two Media Formulations and Two Vented Culture Vessels for Shoot Multiplication and Rooting of Hemp Shoot Tip Cultures

Micropropagation of hemp (Cannabis sativa) is constrained by problems with hyperhydricity and culture decline of microshoots. These problems can be reduced by increasing agar and nutrients in the media during micropropagation stages 1 and 2, respectfully. Performance of microshoots of ‘Abacus’ and ‘Wife’ hemp cultured in Driver and Kuniyuki Walnut medium (DKW) for 15 weeks (6 weeks of stage 1 + 9 weeks of stage 2), with subculturing every 3 weeks during both stages 1 and 2, or in Murashige and Skoog with vitamins medium (MS) for 6 weeks (stage 1) followed by Lubell-Brand Cannabis medium (LBC) for 9 weeks (stage 2), with subculturing every 3 weeks during both stages 1 and 2, was evaluated. In a separate study, microshoot performance of ‘Abacus’ and ‘Wife’ in MS for 3 weeks (stage 1) followed by LBC for 6 weeks (stage 2), with subculturing every 3 weeks, using boxes (Magenta GA-7) with lids featuring a vent with a diameter of 10 mm and a pore size of 0.2 µM or using microboxes (Sac O2 O95/114 + OD95) with lids featuring a filter (Sac O2 #10) were evaluated. Shoot multiplication rate (SMR) and explant height were greater for ‘Abacus’ in LBC than DKW. For ‘Wife’, SMR at 9 weeks was greater in LBC, as LBC provided more nutrients and water than cultures had received in MS initially during stage 1. Culture medium did not influence ex vitro rooting success, which was 75% for ‘Abacus’ and ≥ 90% for ‘Wife’. Microboxes resulted in greater hyperhydricity of shoots and a lower ex vitro rooting percentage than boxes. For cultivars that are highly prone to developing hyperhydricity, like ‘Abacus’, the microboxes were not adequate to control this condition.

Are silver nanoparticles the “silver bullet” to promote diterpene production in Stevia rebaudiana?

S. rebaudiana is a sought after sweetener because of its low-calorie properties. However, the supply of suitable quantities of high quality propagation material is limited by inefficient propagation methods using conventional strategies. In vitro techniques combined with nanotechnology tools offer an attractive alternative not only for improved propagation but also for the stimulation of secondary metabolites which represent the targeted sweetener product for this crop. This report provides an evaluation of silver nanoparticles applied in temporary immersion bioreactors for the abovementioned purpose. Different levels of AgNPs were supplied (0.0—37.5 mg/L) and after 21 d of growth, morphological and biochemical indicators were evaluated. Silver nanoparticles at 25 and 37.5 mg/L decreased shoot multiplication rate, shoot length, and the number of nodes and leaves per shoot compared with the control while no adverse effect was found at the lower tested concentration (12.5 mg/L). Shoot fresh and dry weights also showed statistically significant differences. Regarding the biochemical phenotypes, chlorophyll a, carotenoids and soluble phenolics were increased in plants supplied with 25 mg/L AgNPs, with the latter two indicators suggesting oxidative stress. Interestingly, endogenous levels of diterpenes were significantly increased with the application of 12.5 mg/L AgNPs. It is suggested that AgNPs show potential to act as elicitors to promote the production of diterpenes in stevia but that further work is required to understand the balance between oxidative damage and secondary metabolite production and that optimization of the protocol is required to improve the propagation potential of this strategy.

Impact of Different Sucrose Concentrations on Shoot Multiplication of Papaya (Carica papaya L.) Cultured in vitro

Papaya is a heterozygous plant commonly cultivated by seed but, unfortunately, they are not true to type. Moreover, the hybrid seed varieties like the Red Lady are very highly expensive. Hence, tissue culture techniques offer an alternative method to produce a million clones within a short period and a reasonable price. Thus, the current study aimed to optimize the shoot multiplication rate of papaya (Carica papaya L. cv. Red Lady) in vitro. Five concentrations of sucrose (10, 20, 30, 40, and 50 g.L-1) were applied for the papaya shoot proliferation. Results demonstrated that the 30 g.L-1 sucrose was significantly superior in the rate of shoot numbers (4.1 shoots. explant-1), shoot length (0.90 cm), (2.7 leaves. shoot-1), leaf area (1.40 cm2) and fresh weight (0.192 g) in compared with other sucrose treatments. Whereas, the 40 g.L-1 sucrose treatment was significantly superior in dry weight of shoot compared to the other treatments, which recorded 0.058 g. While the treatment of 10 g.L-1 sucrose recorded the lowest values in shoot numbers, length, and dry weight. Current study conclude that the 30 g.L-1 sucrose is the best concentration treatment that must be used in papaya micropropagation, where it gives the maximum rate of shoot numbers and other morphological traits.

In vitro propagation and improving accumulation of coumarin in Lycium barbarum, a rare plant in the flora of Egypt

BackgroundThe conservation of natural populations of plants is very important for maintaining biological diversity. Lycium barbarum (goji berries) is classified as a very rare plant in the Egyptian flora. Goji berry is a fruit commonly known as ‘super-fruit,’ due to its beneficial constituents and medicinal properties. The aim of this study was to determine the influence of media formula added with various cytokinins on axillary bud induction of L. barbarum from shoot tip. An additional objective of this study was to investigate the effect of 2,4-D, TDZ, zeatin, IBA and NAA on growth and development of the plant through different stages. The present study also aims to improve coumarin accumulation as one of the most active constituents in the plant.ResultsSterilized shoot tips of L. barbarum were cultured without growth regulators on different media formulas: Murashige and Skoog basal medium (MS medium), Gamborg’s medium (B5 medium) and Woody plant medium (WP medium). MS medium gave the best shoot induction. The maximum multiplication rate of shoots was recorded on MS medium supplemented with 225.24 µM 6-benzyl amino purine (BAP). The best callus induction rate from in vitro derived young leaves was obtained on MS medium with 552.60 µM thidiazuron (TDZ). At 176.20 µM TDZ, the maximum shoot proliferation ability was obtained through organogenesis from the callus. MS medium with 220.25 µM TDZ produced the highest number (15 buds) and forming rate (96%) of adventitious buds through direct organogenesis. On MS medium with 304.86 µM naphthalene acetic acid (NAA), 100% rooting was obtained. Almost 95% of the in vitro-generated plantlets were successfully acclimatized. Furthermore, suspension culture containing MS medium with 552.60 µM TDZ and cinnamic acid at a concentration of 763 μM increased coumarin synthesis (1.95-fold), compared to control.ConclusionThe in vitro regeneration of plants via leaf-mediated shoot organogenesis protocol presented in this study is well suited for L. barbarum propagation and produced large quantities of identical planting materials for desert revegetation and pharmaceutical industries. A very effective indirect regeneration method via callus culture provides a potential for this ethnomedicinal plant to be improved by genetic transformation procedures, in addition to mass propagation of elite plants. The cell suspension culture medium with 763 µM cinnamic acid had the highest coumarin accumulation.

Optimal condition for Propagation and Growing of Dendrobium thyrsiflorum

Dendrobium thyrsiflorum is a medicinal orchid that is being gradually destroyed due to over-exploitation. This research focuses on in vitro production of D. thyrsiflorum plantlet under optimal acclimatization conditions. The results of the study showed that the best medium for seed germination was MS basal medium (full-strength MS nutrient plus 100 ml L-1 coconut water; 7 g L-1 agar, 30 g L-1 sucrose, and 0.5 g L-1 active charcoal) supplemented with 60 g L-1 mashed sweet potatoes, which induces 97.8% of seed germination. The shoot was well developed in MS basal medium supplemented with 60 g L-1 mashed sweet potatoes and 0.4 mg L-1 6-Benzylaminopurine (BA) and, corpulent and green in shoot morphology. The shoot multiplication rate was greatest on MS basal medium supplemented with 0.4 mg L-1 BA and 0.4 mg L-1 Kinetin with 4.53 times, and the shoot height was reported at 3.45 cm after 8 weeks of subculture. Further, The shoot was 100% rooting, with an average of 4.51 roots/shoot and 5.34 cm per root in length when the shoot was implanted on MS basal medium plus 60 g L-1 mashed sweet potatoes and 0.75 g L-1 active charcoal. Especially, plantlets after transplanting to the orchid net house reached a 94.8% survival rate on tree fern after 24 weeks. Hence, the results of the study suggested a successful production of D. thyrsiflorum plantlet on the selected media compositions.

Seed germination and in vitro propagation of three threatened endemic South African Aloe species

Aloe modesta Reynolds (endangered), A. peglerae Schönland (critically endangered), and A. reitzii Reynolds (vulnerable) are endemic South African Aloe species valued for their horticultural and/or medicinal value, appearing on the Red List of South African Plants. Propagation is an important step in the cultivation of these species for their conservation. This study examined the effect of temperature (15, 20, 25, and 30°C) and photoperiod (constant dark, 16 h light, and constant light) on seed germination of these three species. The in vitro propagation of A. reitzii was also investigated by examining the effect of different cytokinins [kinetin, 6-benzyladenine (BA), meta-topolin (mT), and meta-topolin riboside (mTR)] on shoot multiplication. Germination percentage and mean germination rate of A. modesta were significantly inhibited when the seeds were incubated at a low temperature (15°C) in comparison to incubation at other temperatures (20 – 30°C). An increase in temperature significantly decreased mean germination time (MGT), as well as increased seedling chlorophyll and carotenoid contents in A. peglerae and A. reitzii. A reduction in germination speed, evident in increased MGT was observed in the three species when seeds were incubated under constant light and 16 h light as compared to constant dark. Medium with 5.0 µM meta-topolin produced the highest A. reitzii shoot proliferation (16 shoots) per shoot-tip explant after eight weeks of culture. This shoot multiplication rate translates to a potential production of 1 118 481 shoots per shoot-tip explant per annum, based on a geometric progression with six possible multiplication cycles per annum. Aloe reitzii shoots produced from BA and kinetin treatments had a high flavonoid content whereas all mT and mTR treatments gave reduced flavonoid content. Seed and/or in vitro propagation protocols of these threatened endemic Aloe species established in this study can be employed in the biodiversity conservation of Aloe species for their sustainable use.

Micropropagation from Inflorescence Nodal Segments of Phalaenopsis and Acclimatization of Plantlets Using Different Substrates

Phalaenopsis is an orchid genus of great economic value in world floriculture. In vitro clonal propagation is the only large-scale feasible method for Phalaenopsis propagation, but it is difficult because of the low multiplication rate. The aim of this study was to evaluate the effect of types and concentrations of N6-benzyladenine (6-BA) and gibberellic acid (GA3) on the in vitro multiplication of shoots from inflorescence nodal segments (INS) of Phalaenopsis hybrids. INS with one axillary bud were inoculated in New Dogashima Medium with different combinations of BA and GA3. The results show that the treatment containing 1.0 mg L−1 BA and 1.5 mg L−1 GA3 showed the higher percentage of live inflorescence segments (71.48%) and a number of shoots (1.68 shoots/INS). The highest 6-BA concentration (4 mg L−1) tested in this study resulted in the best shoot multiplication rate (4.3). Contamination and browning of the INS tissues were the main difficulties identified for clonal propagation of Phalaenopsis. Successful in vitro rooting occurred on half-strength Murashige and Skoog medium (100%), and acclimatization (100%) was obtained independent of the substrates. However, the best gains in number of roots, leaves, chlorophyll content, and fresh weight of plantlets were achieved using vermiculite.

Polyethylene glycol mediated improved shoot proliferation, foliar morpho-anatomy, and rooting of micropropagated shoots of Spathoglottis plicata Blume

In vitro propagation is extremely important for the rapid multiplication of a wide range of orchids. This study aimed to investigate the effects of polyethylene glycol (PEG) on morpho-anatomical improvements and ex vitro development of Spathoglottis plicata, a terrestrial orchid. Various concentrations of PEG (100-500 mg L−1) were incorporated with the optimized medium composition [Murashige and Skoog's medium with 2.0 mg L−1 6-benzylaminopurine (BAP) and 0.25 mg L−1 indole-3-acetic acid (IAA)] to study the morpho-anatomical developments of cultures. Results showed that PEG at 300 mg L−1 in the nutrient medium had a significant effect on the development of plantlets with superior morphometric and anatomical characteristics. It improved the rate of shoot multiplication (83 shoots), shoot length (10 cm), fresh weight (4.0 g), and dry weight (1.5 g) of shoots per culture vessel than the control experiment (devoid of PEG). Light microscopic analysis revealed that PEG-derived leaves possessed improved leaf thickness, differentiation in dermal, ground, and vascular tissue systems than the leaves developed without PEG in the medium. Such shoots were responded significantly better during rooting (11.0 roots with 6.2 cm length) and acclimatization stages. Start Codon Targeted (SCoT) markers analysis confirmed the genetic uniformity of the regenerates with the mother plant. The improvements in morpho-anatomical structures of in vitro raised plantlets due to PEG-induced mild drought stress in ground orchid S. plicata could be a useful tool for its large-scale production and survival success in the field.

\(\textit{In vitro}\) shoot multiplication and the content of huperzine a in in vitro cultured \(\textit{Huperzia serrata}\) (Thunb. Ex Murray) Trevis plants

Huperzia serrata (Thunb. Ex Murray) Trevis belonging to the Lycopodiaceae family contains the main active ingredient Huperzine A (HupA), which is effective in the treatment of dementia. However, H. serrata in nature contains a low content of HupA. Additionally, this plant grows very slowly and is overexploited, thereby leading to the risk of extinction. In this study, we used the in vitro shoot of H. serrata to induce shoot multiplication by tissue culture method and quantify the content of HupA in the in vitro cultured plant by High-Performance Liquid Chromatography (HPLC). The suitable nutrient medium for inducing shoot multiplication was 1/4 MS medium supplemented with 1 mg/L of kinetin. In this medium, the shoot multiplication rate was as high as 77.33%, the number of shoots/shoot multiplication was 6.02, and the shoot height was 1.61 cm. Based on the HPLC method, the content of HupA in the in vitro cultured plant was 300 µg/g. The content of HupA in the cultured H. serrata in Vietnam is reported for the first time in our study. This study shows in vitro shoot multiplication is a promising method for propagating H. serrata and can produce the cultured H. serrata with a high content of HupA. These findings contribute to conserving and preserving the gene source of H. serrata species, and at the same time, enhancing the content of HupA in the cultured H. serrata.

In vitro propagation of Bambusa balcooa by plant tissue culture technique

Bambusa balcooa is a common plant grown in Bihar, Orissa, Jharkhand and Uttarakhand states of India having good cultural importance. The present experimentation on nodal explants of B. balcooa on MS media supplement under particular culture conditions has shown shunted growth in 30 days. Shoot tip explants of B. balcooa seedlings produced multiple shoots on MS medium supplemented with different plant growth regulators (PGRs) individually and in combination. Shoot tip explants of B. balcooa requires 30 days to initiate shoots. Among the three cytokinins tested, BAP was selected as the most suitable hormone to induce shoot multiplication. The highest shoot multiplication is found by incorporation with BAP (1.5 µg/L and 2 µg/L). The shoot multiplication rates are good with shoot length 2.5±0.3 cm and 3.9±0.4 cm with the highest and maximum shoot generation. The shoot multiplication rate is moderate with a shoot length of 2.00±0.5 cm. Multiplication potentiality was observed in the cluster having more than 2–3 shoots. The best period for recycling multiplying shoots is 2–3 weeks in the old culture. Delaying of the sub-culturing period resulted in gradual browning of the shoots. The sub-culturing period was recorded as the most crucial factor for obtaining the optimal and desired level of regeneration of shoots. The well-grown propagules in the present experimentation were shown successful regeneration. Hence, the experiment concluded with the best growth pattern observed in media containing auxins like BAP and cytokinins like kinetin.

Changes in Endogenous Phytohormones of Gerbera jamesonii Axillary Shoots Multiplied under Different Light Emitting Diodes Light Quality.

Light quality is essential in in vitro cultures for morphogenesis process. Light emitting diodes system (LED) allows adjustment as desired and the most appropriate light spectrum. The study analyzed the influence of different LED light quality on the balance of endogenous phytohormones and related compounds (PhRC) in in vitro multiplied axillary shoots of Gerbera jamesonii. Over a duration of 40 days, the shoots were exposed to 100% red light, 100% blue light, red and blue light at a 7:3 ratio with control fluorescent lamps. Every 10 days plant tissues were tested for their PhRC content with the use of an ultra-high performance liquid chromatography (UHPLC). Shoots’ morphometric features were analyzed after a multiplication cycle. We identified 35 PhRC including twelve cytokinins, seven auxins, nine gibberellins, and seven stress-related phytohormones. Compounds content varied from 0.00052 nmol/g to 168.15 nmol/g of dry weight (DW). The most abundant group were stress-related phytohormones (particularly benzoic and salicylic acids), and the least abundant were cytokinins (about 370 times smaller content). LED light did not disturb the endogenous phytohormone balance, and more effectively mitigated the stress experienced by in vitro grown plants than the fluorescent lamps. The stress was most effectively reduced under the red LED. Red and red:blue light lowered tissue auxin levels. Blue LED light lowered the shoot multiplication rate and their height, and induced the highest content of gibberellins at the last stage of the culture.

In Vitro Technologies for American Chestnut (Castanea dentata (Marshall) Borkh) Conservation.

American chestnut (Castanea dentata), a native species of eastern North America, is an economically important deciduous hardwood tree that has been designated as endangered in Canada. The population of American chestnut trees has dwindled significantly across Southern Ontario due to chestnut blight and many of the surviving trees continue to show blight disease symptoms. American chestnut requires efficient strategies for propagation and preservation for species recovery. The objective of this study was to develop a long-term plant conservation program using micropropagation and cryopreservation protocols. An in vitro technology using a liquid-based temporary immersion system (TIS) was developed for micropropagation of American chestnut. The highest rate of shoot multiplication was observed in cultures grown in the DKW (Driver and Kuniyuki 1984) basal medium supplemented with 2.2 µM 6-benzylaminopurine and 1.0 µM gibberellic acid. More than 95% of proliferated microshoots, about 40–50 mm in size, developed roots after 30 days of culture within bioreactor vessels containing DKW basal medium supplemented with 15 µM 3-Indolebutyric acid. Rooted plantlets transplanted to the greenhouse had a survival efficiency of 82% after one month of growth. The cryopreservation protocol for germplasm preservation was developed through droplet vitrification of shoots. Optimal regeneration of shoot tips occurred from explants precultured on stepwise concentrations of sucrose and subsequent dehydration in PVS3 for 30 min. Cryopreserved shoot tips were regenerated to whole plants using pre-optimized conditions of micropropagation. This study confirms the potential of TIS for micropropagation in ex situ conservation and reintroduction of endangered American chestnuts and possibly other woody plant species.

Synergistic effect of benzylaminopurine and meta-Topolin combination for micropropagation of gerbera ‘Pink Melody’

ABSTRACT Gerbera (Gerbera jamesonii Bolus ex Hook. F.; Asteraceae), is one of the most economically important ornamental plants due to its aesthetic value. In the present study, we established a micropropagation method for the large-scale production of quality planting material of gerbera ‘Pink Melody’. Eighty-six percent of the capitulum explants produced adventitious shoots (15.44 ± 0.34 shoots per capitulum) on Murashige and Skoogs (MS) medium containing 2 mg L−1 6-benzylaminopurine (BAP) after six weeks of incubation. The highest shoot multiplication rate (17 shoots per explant) was obtained on MS medium supplemented with BAP and meta-Topolin (each at 2 mg L−1) after eight weeks. The micro-shoots were successfully rooted (91.35%) on half-strength MS medium containing 2 mg L−1 indole-3-butyric acid (IBA) within four weeks. The micropropagated plantlets were acclimatized with a 97.5% survival rate and produced flowers with no visible morphological aberrations.

Coconut water promotes In vitro shoot multiplication rate from strawberry (Fragaria × Ananassa) nodal segments

In vitro micropropagation of strawberry cv. Chandler was carried out from nodal segments. Among different disinfectants used, treatment of segments with mercuric chloride (0.1% w/v) for 5 seconds showed 90% establishment of segments. Among different media tested, solid media containing Benzyl Amino Purine (0.2 mg/L), Indole Acetic Acid (0.2 mg/L), Kinetin (0.3 mg/L), Table sugar/Sucrose (30 g/L), Coconut water (150 ml/L), Agar (8 g/L) were found to be most effective for shoot multiplication, and root formation occurred within two weeks. The liquid medium containing BAP (0.2 mg/L), IAA (0.2 mg/L), Kin (0.3 mg/L) and Sucrose (30 g/L) was found to be at par with the above medium with respect to shoot multiplication, and root formation occurred in 10 days on this medium. Plants grown in leguminous soil showed more than 90% survival in the screen house.