Rapid Mass Propagation Research Articles

Vegetative propagation of Phytolacca acinosa Roxb. by rhizome cuttings: a step towards conservation and cultivation approach

IntroductionPhytolacca acinosa Roxb. is a highly valuable multipurpose herb native to the Himalayan region. Unsustainable harvesting of this species due to its diverse uses has resulted in a rapid decline in its population across natural habitats, thereby necessitating its propagation and conservation. To overcome this challenge, the potential of P. acinosa rhizomes for ex situ regeneration was evaluated.MethodsThe current study aims to develop a standard propagation protocol for P. acinosa. Rhizome cuttings derived by splitting whole rhizomes were used to study the effect of various hormones and soil compositions on their sprouting and growth performance.ResultsSoil compositions SC10 and SC5 consisting of soil, sand, pebbles, and vermicompost (1:1:1:1) and soil, sand, and vermicompost (1:1:1), respectively, were the most suitable compositions for the optimum growth of this species. The rhizome segments treated with GA3 (150ppm) induced the highest sprouting percentage (91.67%), with a minimum sprouting time of 23.25 days. The maximum root length (9.25 cm), shoot length (16.5 cm), and leaf number (11.25) were recorded for GA3 (150ppm) treated rhizome cuttings.ConclutionsOverall, the results of the present study helped in establishing a cost-effective, rapid, efficient, and simple mass propagation method for the target species. The results of this study will serve as a guide for the large-scale cultivation, effective conservation, and sustainable utilization of this economically valuable medicinal herb.

In vitro Callus Regeneration and Chlorophyll Content Estimation in Glycine max (L.) variety from Uttarakhand, India

Soybean (Glycine max) is considered one of the most substantial produced crops across the globe because of its high nutritional value. It is a great alternative for lactose-intolerant patients and vegans to fulfil their daily protein requirements. Soybean has various varieties depending upon the colour of their seed coat. In the past few years, the consumption of Soybean has increased which demands higher production, better yield, and better seed quality. Conventional propagation methods fail to fulfil such demands. The alternate method of plant tissue culture ensures rapid mass propagation, better yield, and quality of the plants. However, the technique is often beset with challenges of low-field performance of tissue culture-raised plants due to defective chloroplast machinery. The present study investigates the effect of various plant growth regulators (PGRs) on in vitro propagation of soybean cultivars from different regions of Uttarakhand, India, and their effect on chlorophyll content in the regenerated tissues.

Performances of plantlets from selected cassava (Manihot esculenta Crantz) genotypes under Semi - Autotrophic Hydroponics (SAH) using different substrates

The cassava seed system faces challenges due to limited seed stock caused by a slow propagation rate and a lengthy growth period. Conventional methods lacking phytosanitary guarantees further compound these issues. To address these limitations, the Semi-Autotrophic Hydroponics (SAH) technology was adopted for the rapid mass propagation of healthy, disease-free cassava plants. This research aimed to evaluate the performance of cassava planting materials using different substrates for stem-cutting multiplication in the laboratory at the IITA Kalambo research station in DR Congo. The experiment followed a split-plot design of five replications of three consecutive sub-culture periods lasting four weeks each. Four different genotypes: IB961089A, MM060083, Nase14, and Albert28 as the main plots, with four different substrates: KlasmannTS3, Vermiculite, Local Peat, and Sawdust as the sub-plots, were laid out. Cuttings were placed in 500-ml substrate-filled boxes and watered weekly with a 100-ml Miracle-Gro solution. Data were collected on survival, height, leaf and internode numbers, and cutting numbers at the end of each subculture period. The data were analysed using ANOVA in R software. The Fisher's Least Significant Difference (LSD) test was utilised to separate means when significant differences among treatments were present (p <0.05). The results showed that survival was primarily influenced by the substrate used, with KlasmannTS3 demonstrating the highest rate, exceeding 90%. Significant differences (p < 0.05) among genotypes for survival rate and performance parameters mentioned above (p < 0.001) were observed. Similarly, there were significant differences (p < 0.001) among substrates for survival rate and performance parameters. Furthermore, the interaction between genotype and substrate significantly (p < 0.001) affected performance parameters. MM060083 performed the best across all traits. KlasmannTS3 was the superior substrate and had the highest average cutting number regardless of genotype, with a notable increase of 292% from 20 to 58.4 cuttings, representing a ratio of 1:3 within three months. Sawdust had the lowest multiplication rate, with a 5% decrease. The superior performance of KlasmannTS3 was attributed to its rapid growth and favourable properties. The interaction between MM060083 and KlasmannTS3 consistently showed the highest number of cuttings (70.4). Sawdust consistently showed poor growth performance, regardless of genotype. The study concludes that the SAH offers the potential for rapid multiplication of disease-free cassava planting materials in reduced space and time.

Rapid mass propagation of endangered valuable medicinal plant <em>Salacia chinensis</em> L. and GC-MS/LC-MS analysis of active compounds produced in callus and leaf extracts

A protocol for indirect organogenesis of Salacia chinensis was established. Normally S. chinensis is propagated through seeds. However, due to the difficulty to obtain uniform plants in a short time period by seed germination, micro-propagation is a possible alternative method. For the micro-propagation, media with different concentration combinations of cytokinins and auxins were used to induce callus formation in three explants types: leaf segment, nodal segment and seeds. The rate of recurrence of callus formation from leaf on Murashige and Skoog (MS) basal medium supplemented with thidiazuron (TDZ) 0.5mg and TDZ 1mg was 100% and the maximum percentage of shoot let development from nodal segment on MS basal medium supplemented with BAP 6-(benzylamino purine) 3.5+ indole3-butyric acid (IBA) 1 mg/l was 78.3% when compared to other plant growth regulators (PGR) combinations used. The highest shoot regeneration response (85%) and the determined shoots (12.33 ± 0.33%) per callus were attained from leaf explants on MS medium containing 1-napthaleneacetic acid (NAA) 1mg/l + BAP 0.5 mg/l. The seeds showed highest percentage of shoot formation on MS medium supplemented with BAP 2mg + indole 3-acetic acid (IAA) 2mg/l and BAP 2mg + isopentenyl adenine (2iP) 2mg/l. Highest root formation (70±1.3%) was found in shoot regenerated using leaf segment on MS medium supplemented with IBA 0.5mg. The gas chromatography mass spectrometry (GC-MS) analysis of methanolic extract of callus showed more compounds at higher percentage. HPLC-MS analysis of methanolic extract of callus showed higher concentration of Mangifera than in leaf extracts are reported for the first time.

Micropropagation of the potential blueberry rootstock—Vaccinium arboreum through axillary shoot proliferation

Vaccinium arboreum is adapted to soils with low organic matter and high pH. It is considered as a potential blueberry rootstock. The objective of this research was to establish a rapid mass propagation protocol of V. arboreum through axillary shoots proliferation with minimum somaclonal variation. Nodal segments from cuttings were cultured on Driver and Kuniyaki walnut medium (DKW) with different cytokinins for initial culture establishment. DKW, woody plant medium (WPM), and Long and Preece medium (LP) were compared for best shoot proliferation. Zeatin (ZT) concentrations from 0 to 4.56 μM were evaluated for optimizing the concentration. Both in vitro and ex vitro rooting were evaluated. The pH tolerance of in vitro shoots was investigated by adjusting the media pH from 5 to 8.5. Best shoot proliferation was achieved by LP medium supplemented with 2.28 μM ZT and 0.05 μM indole-3-butyric acid (IBA). In vitro shoots proliferated best on medium pH 5, though the shoots could survive on pH 8. All the micropropagated shoots successfully rooted by ex vitro rooting after dipping with auxin. All rooted plantlets acclimatized and grew well in greenhouse. Micropropagation through axillary shoots proliferation is an efficient method to propagate true-to-type V. arboreum in large number for future use as rootstock.

True-to-type micropropagated plants of para rubber (Hevea brasiliensis Müll. Arg.) via somatic embryogenesis

Plant micropropagation via somatic embryogenesis is a powerful technique for rapid mass propagation, especially in para rubber (Hevea brasiliensis Müll. Arg.). However, somaclonal variations are the major limitation of this process. To date, DNA fingerprinting, i.e., RAPD (Randomly Amplified Polymorphic DNA), Star Codon Targeted (SCoT), and SSRs (Simple Sequence Repeats), is one of the most successful technologies to detect the genetic fidelity in the somatic embryos. The aim of present study was to induce somatic embryos from inner integument explants of para rubber cv. ‘RRIM 600’ at different developmental stages and subsequent acclimatization and transplantation (under greenhouse and field conditions) of the propagated seedlings. The genetic stability of the plants derived from somatic embryos was also analysed in comparison to the mother plant using RAPD, SCoT and SSRs markers. Somatic embryos derived from inner integuments of 5-week-old immature seeds after pollination were more efficient than older and younger seeds. In addition, para rubber mother plants cv. ‘RRIM600’ and plants derived from somatic embryogenesis demonstrated the same pattern of DNA fragments, as confirmed by three PCR-based techniques, RAPD, SCoT and SSRs, whereas these in the pattern were different from ‘RRIT 226’, ‘PB 235’, ‘PB 251’, ‘PB 255’ and ‘BMP 24’. Interestingly, T2 plant was found to possess somaclonal variations when compared with mother plant. Based on the results, we confirm that the plants derived from somatic embryogenesis of para rubber cv. ‘RRIM 600’ were true-to-type to that of ‘RRIM 600’ master stock.

The Prospect of Physiological Events Associated with the Micropropagation of Eucalyptus sp.

Micropropagation is a reliable technique in biotechnology and genetic engineering domain, which has been widely applied for rapid mass propagation of plants in vitro condition. Through micropropagation techniques, reproduction of plants can be attained from different explants using organogenesis and somatic embryogenesis. Over the decades, micropropagation techniques have offered tremendous potential for forest tree improvement. Eucalyptus is a woody plant species recalcitrant to in vitro culture. In general, the micropropagation of Eucalyptus culture processes and the genotype, environment surroundings, and age of explants in culture media is frequently linked with the occurrence of micropropagation variation. In the current review paper, an update of the most important physiological and molecular phenomena aspects of Eucalyptus micropropagation was linked to the most profound information. To achieve the mentioned target, the effect of plant growth regulators (PGRs), nutrients, other adjuvant and environmental features, as well as genetic interaction with morpho- and physiological mechanisms was studied from the induction to plant acclimatisation. On the other hand, important mechanisms behind the organogenesis and somatic embryogenesis of Eucalyptus are discussed. The information of current review paper will help researchers in choosing the optimum condition based on the scenario behind the tissue culture technique of Eucalyptus. However, more studies are required to identify and overcome some of the crucial bottlenecks in this economically important forest species to establish efficient micropropagation protocol at the industrial level.

A Study on In Vitro Propagation Possibilities of Some Clone Rootstocks of Prunus Species

In vitro techniques which is one of the modern breeding methods offer alternative methods to meet the growing food demands in parallel with the increasing world population by allowing rapid and intensive mass propagation of varieties and rootstocks in fruit cultivation under laboratory conditions. Clone rootstocks which are economically important and early yielding with maximum yield per unit area are preferred in fruit production. In this study, the propagation possibilities of Marianna GF 8/1, Myrobolan B, Myrobolan 29-C and St Julien A clone rootstocks used in the production of stone fruit species were investigated. In this study, shoot tips were used as explants. MS (Murashige-Skoog) media was used as the propagation media and 30 g L-1 sucrose and 7 g L-1 agar were added. The shoot tips were planted into the media and then placed in the temperature and light controlled culture chamber. Samples were placed to the subculture at 1-month intervals. Explant length, number of leaves and number of tillings were observed every 20 days. Nine different MS media were tested during the shoot growth stage. The best propagation was obtained in the 1 mg L-1 BAP + 0.5 mg L-1 IBA + 0.25 mg L-1 GA3 media. Eight different MS media were tested during the rooting stage. The best rooting was achieved in a media containing 1/2 MS + 2 mg L-1 IBA. Keywords: Marianna GF 8/1, Myrobolan B. Julien A, Myrobolan 29-C, micropropagation, plant growth regulators. DOI: 10.7176/JSTR/5-9-10

In vitro Rapid Mass Propagation from Leaf Culture of <i>Drosera rotundifolia</i>

This study was conducted to establish a rapid mass propagation system by leaf culture of Drosera rotundifolia, a representative insectivorous plant that grows naturally in Korea. We evaluated the effects of explants [leaf blade, leaf blade (cut-square), petiole, leaf blade+petiole, leaf blade+petiole (center-cut)], media strength [1/4 MS (Murashige and Skoog), 1/2 MS, 1MS, and 2 MS], culture methods (liquid and solid media), culture condition (1 week under light after 4 weeks under dark, 3 weeks under light after 2 weeks under dark and 5 weeks under light) and cytokinin (BA, kinetin) on shoot and plant regeneration. In the treatment by solid medium, of shoots were the highest (100%, 1.8 shoots per explant) in 1/4 MS and 1/2 MS with leaf blade (cut-square), and leaf blade+petiole with 100% and 1.6 - 1.8 shoots per explant. In the treatment by liquid medium, plant regeneration rate was shown to be 100% in 1/2 MS with leaf blade (cut-square), and number of shoots was high f rom 2.6 - 2.7 in 1/4 MS and 1/2 MS, respectively, both with leaf blade (cut-square) and leaf blade+petiole (center-cut). Comparing solid to liquid medium, the result of culturing of liquid medium for five weeks is better than that of solid medium for eight weeks, which is deemed to be efficient for plant regeneration in D. rotundifolia. In culture conditions, the highest number of shoots was 3.5 - 3.6, respectively, in 1/4 MS with 1 week under light after 4 weeks under dark and 3 weeks under light after 2 weeks under dark. In conclusion, the growth of D. rotundifolia, 1/4 MS with 3 weeks under light after 2 weeks under dark are considered the best for the in vitro growth of D. rotundifolia. In this experiment, the shoot and root growth were found to be better in MS medium without cytokinin rather than with cytokinin.

Effect of phytohormones on rapid In vitro propagation of dendrobium thyrsiflorum Rchb.f.: An endangered medicinal orchid

Pharmacognosy Magazine,2018,14,58,495-500.DOI:10.4103/pm.pm_155_18Published:November 2018Type:Original ArticleAuthors:Leimapokpam Tikendra, Thoungamba Amom, and Potshangbam Nongdam Author(s) affiliations:Leimapokpam Tikendra, Thoungamba Amom, Potshangbam Nongdam Department of Biotechnology, Manipur University, Imphal, Manipur, India Abstract:Background: Dendrobium thyrsiflorum is highly vulnerable to extinction in its natural habitats due to overexploitation for the medicinal and horticultural purposes. The poor seed germination in a natural condition and slow plant production through vegetative approach make in vitro micropropagation techniques an ideal choice for effective plant propagation. There are very limited reports on the in vitro culture of this orchid. Objective: The objective of the study is to establish efficient in vitro regeneration protocols for rapid mass propagation of D. thyrsiflorum. Materials and Methods: Five-month-old undehisced capsule was surface sterilized with 0.4% mercuric chloride. To study the influence of growth regulators on in vitro development of shoots and roots, the seeds obtained from the capsule were inoculated on freshly prepared Mitra medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP), kinetin (KN), indole -3-butyric acid (IBA), indole-3-acetic acid (IAA), and 1-naphthyl acetic acid (NAA). Results: The KN either singly or with IAA produced more effective shooting than BAP at similar concentration and combination. Maximum shoot production (3.83 ± 0.48) was achieved in medium fortified with 1.0 mg/L KN and 2.5 mg/L IAA. IAA was the most influential among auxins tested in promoting rooting and shooting. Prominent shoot development (3.04 ± 0.73) and maximum root formation (6.52 ± 0.37) were noticed in medium supplemented with 2.0 mg/L IAA. The regenerated plantlets were subsequently hardened with 91% survival rate in greenhouse and field condition. Conclusion: The present investigation revealed the possibility of developing effective and reproducible in vitro regeneration protocols for rapid propagation of D. thyrsiflorum. The established in vitro protocols can be employed for fast regeneration of the orchid for useful phytochemical extraction without depending on the already depleted natural population. Keywords:Auxin, Cytokinin, Dhanzyme gold, Propagation, ProtocormView:PDF (1.74 MB)

High frequency direct shoot organogenesis of leaf explants and a comparative evaluation of phytochemicals, antioxidant potential of wild vs. in vitro plant extracts of Lysimachia laxa.

The present studies were attempted to develop direct shoot organogenesis from in vitro grown leaf explants of Lysimachia laxa and comparative evaluation of phytochemical and antioxidant potential of in vitro raised and wild plants extracts. The fresh leaves of this species are used for deworming gastrointestinal worm infection in traditional medicine. Overexploitation of this species and poor regeneration has led to rapid decline in wild population, therefore, present investigation was attempted to develop an efficient rapid mass propagation protocol for this species. Our result showed significantly (P<0.05) high adventitious shoot proliferation of 17.21±0.24 number per leaf explants cultured in Murashige and Skoog medium fortified with 1.25mgL-1 thiadizuron and 1.0mgL-1 α-naphthalene acetic acid. Further enhancement was achieved through elongation medium fortified with 1.0mgL-1 6-benzylaminopurine by average shoot number of 31.1±0.80 and length of 5.96±0.13cm. Murashige and Skoog medium fortified with 0.50mgL-1 Indole-3-acetic acid showed high rooting induction (100%) with average root number of 11.70 and length 7.35cm. All rooted plants were successfully acclimatized in greenhouse and transferred to field condition with a survival rate of 97%. The contents of phenolic and flavonoid were higher in in vitro raised plant in compared to wild plant extracts. Antioxidants assay showed high radical scavenging activity of IC50 1.61±0.07mg dry material and reducing power of 49.79±0.11mg/g ascorbic acid equivalent by aqueous methanol extracts of in vitro raised 3-months-old plants in compare to the wild plants. The present protocol is a viable option for pharmaceutical or nutraceutical industries for sustainable utilization of L. laxa with enhanced of phytochemical and antioxidant potency which is not reported elsewhere.

Enhanced production of berberine in In vitro regenerated cell of Tinospora cordifolia and its analysis through LCMS QToF

Tinospora cordifolia is a prioritized medicinal plant and having an immense medicinal importance especially in Indian medicinal system. But this plant needs a regeneration protocol for its rapid propagation. An efficient regeneration protocol was developed for T. cordifolia using nodal explants. High frequency of multiple shoot formation was induced when the nodal segments were cultured on MS medium supplemented with BAP (1.0 mg L−1) and 2-iP (0.5 mg L−1). The highest mean number of shoots per nodal explant (7.9 ± 0.45) with highest shoot length (9.3 ± 0.48 cm) and 86% response were achieved on this media and hormonal concentration. The optimum rooting was obtained on ½ strength of MS medium augmented with IBA (0.5 mg L−1) with 8.3 ± 0.46 cm root length and 89% response. Micropropagated plantlets were found to be identical with the mother plant when clonal fidelity of these plantlets were analyzed with inter simple sequence repeat (ISSR) marker. The berberine content was analyzed through LCMS QToF and the highest amount was found in in vitro callus (19.8 µg/gm) followed by stem (9.3 µg/gm) and leaves of field-grown plants (8.4 µg/gm). Further, presence of berberine was confirmed by ESI–MS spectra with protonated molecular ions ([M + H]+) at m/z 336. Furthermore, MS–MS fragmentation pattern confirmed for the presence of berberine in both the samples. Both the spectra (standard and samples) showed common peaks for berberine in the form of protonated molecular ions ([M + H]+) at m/z 320, m/z 304, m/z 292, m/z 278 in MS/MS mode. The study revealed that developed protocol is potent for rapid mass propagation of this plant species with high accumulation of important secondary metabolite berberine.

Addressing Challenges to Enhance the Bioactives of Withania somnifera through Organ, Tissue, and Cell Culture Based Approaches.

Withania somnifera is a highly valued medicinal plant in traditional home medicine and is known for a wide range of bioactivities. Its commercial cultivation is adversely affected by poor seed viability and germination. Infestation by various pests and pathogens, survival under unfavourable environmental conditions, narrow genetic base, and meager information regarding biosynthesis of secondary metabolites are some of the other existing challenges in the crop. Biotechnological interventions through organ, tissue, and cell culture provide promising options for addressing some of these issues. In vitro propagation facilitates conservation and sustainable utilization of the existing germplasms and broadening the genetic base. It would also provide means for efficient and rapid mass propagation of elite chemotypes and generating uniform plant material round the year for experimentation and industrial applications. The potential of in vitro cell/organ cultures for the production of therapeutically valuable compounds and their large-scale production in bioreactors has received significant attention in recent years. In vitro culture system further provides distinct advantage for studying various cellular and molecular processes leading to secondary metabolite accumulation and their regulation. Engineering plants through genetic transformation and development of hairy root culture system are powerful strategies for modulation of secondary metabolites. The present review highlights the developments and sketches current scenario in this field.

In vitro propagation and genetic fidelity analysis of alginate-encapsulated Bacopa monnieri shoot tips using Gracilaria salicornia extracts

The scope of the present study is to investigate the effect of plant growth regulators and seaweed liquid extracts (SLEs) on in vitro regeneration of the Ayurvedically important memory plus plant Bacopa monnieri using shoot tips. Sodium alginate-encapsulated shoot tips were cultured on Murashige and Skoog (MS) medium containing 6-benzylaminopurine (BAP), kinetin, zeatin, 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA), individually for shoot initiation. The effect of Gracilaria salicornia and Kappaphycus alvarezii SLEs (20–80 %) for shoot induction and regeneration using shoot tips was also studied. MS medium containing 1.0 mg L−1 BAP and 0.5 mg L−1 NAA showed highest multiple shoot induction frequency 95.8 %, mean number 147.8, and mean length 12.5 cm of shoots where G. salicornia extract at 60 % showed 85.9 % multiple shoot induction with 145.6 shoots and mean length 12.3 cm after 5 weeks of incubation. An effective rooting frequency 84.1 % was observed on half-strength MS medium containing 0.1 mg L−1 IAA and where 25 % of G. salicornia extract showed 82.2 % of rooting after 1 week of incubation. In vitro-rooted plants were acclimatized in soil cups and maintained in greenhouse with natural conditions. In vitro-propagated and mother plants were analyzed using random amplified polymorphic DNA markers reveled that no genetic variation between control plants and in vitro-regenerated plants using SLEs. These results showed that G. salicornia extracts can be used as PGRs for in vitro propagation of B. monnieri and it is a simple, rapid mass propagation method.

Micropropagation of &lt;i&gt;Genista aetnensis&lt;/i&gt; [(Raf. ex Biv.)DC

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

Micropropagation of Genista aetnensis [(Raf. ex Biv.)DC

Genista aetnensis [(Raf. ex Biv.)DC] is a large deciduous shrub or small tree native to the Italian islands of Sardinia and Sicily. Being winter hardy and characterized by high plasticity in altitude and ecology, the species is grown in gardens and landscaping, both for flower and for its attractive shape. Genista species are generally propagate by seed or semi-hardwood cuttings. In this report an efficient in vitro technique for propagation of G. aetnensis was investigated. Multiple shoots were induced on nodal segments of a mature plant of Genista aetnensis. The Murashige and Skoog medium, augmented with different concentrations of N-6-benzyladenine either singly or in combination with indole-3-acetic acid, as potential medium for shoot multiplication by nodal segments was tested. In the following experiment equal molar concentrations of four cytokinins (2-isopenthenyladenine, kinetin, zeatin and N-6-benzyladenine) were tested for ability to induce axillary shoot development from single node stem segments. The highest rate of axillary shoot proliferation was induced on the medium supplemented with 0.44 µM BA. Growth regulator requirements for shoot proliferation in G. aetnensis were satisfied by BA alone. Explants were divided, subcultured and continued to proliferate shoots. A proliferation rate of 3.5 shoots per single node explants every four weeks occurred. Seven indole-3-acetic acid concentrations (0, 0.23, 0.45, 0.91, 1.82, 3.64 or 7.29 µM) were tested to determine the optimum conditions for in vitro rooting of microshoots. The highest rooting percentage was obtained with indole-3-acetic acid at 3.64 mM (57%). Eighty percent of the in vitro rooted plantlets were successfully established in soil. This micropropagation system of G. aetnensis based on axillary shoot development from nodal segments followed by in vitro rooting should be preferred for rapid and efficient mass propagation of selected clones and could represent an alternative method to sexual and conventional asexual propagation.

In vitro Rapid Mass Propagation by Inverted Planting of Corm Explant in Domestic Breeding Freesia

In vitro Rapid Mass Propagation by Inverted Planting of Corm Explant in Domestic Breeding Freesia

Asymbiotic Germination of Immature Seeds in an Ornamentally Important'Fox-tail ’Orchid,Aerides multifloraRoxb.

Present paper deals with asymbiotic ger-mination potential of immature seeds in Aeridesmultiflora Roxb. for its rapid mass propagation in vitro. Seeds were inoculated on Potato Dex-trose Agar (PDA) medium with and without growth adjuncts (CH, Casein Hydrolysate; P, Pep-tone, YE, Yeast Extract; each 0.1%) and activated charcoal (AC; 0.2%). Seed germination initiated after 1.20±0.42 weeks (wks) on CH supplement-ed medium. The percent germination response showed significant differences in majority of combinations used; it was lowest (48.00±2.16) in basal medium and highest (99.50±0.85) in medi-um supplemented with CH and AC. Protocorm formation started after 4.15±0.48 wks of cultur-ing on CH and AC containing medium. First leaf and first root differentiated after 7.30±0.82 and 14.00±0.67 wks of culturing respectively on me-dium supplemented with P and AC. Seedlings with 3–4 leaves and 2–3 roots were obtained af-ter 18.30±0.67 wks. The medium supplemented with CH and AC proved to be the best for seed germination, protocorm formation and multipli-cation, and that containing P and AC for early leaf and root differentiation, and seedling devel-opment. The seedlings were subjected to hard-ening and 72.56±3.18% of them survived under greenhouse conditions.

In vitro pseudobulb based micropropagation for mass development of Cymbidium finlaysonianum Lindl.

An efficient protocol for micropropagation using the axenic pseudobulb derived from in vitro germinated seedlings of Cymbidium finlaysonianum Lindl was developed. The immature seeds were germinated on MS0 basal medium for seedling development. In vitro grown seedlings responded well and stimulated pseudobulb formation with benzylamino purine (BAP) 1.0 mg/L + α-naphthalene acetic acid (NAA) 0.5 mg/L on MS medium. For multiple shoot induction, the axenic pseudobulb segments excised from 8 month old seedlings were cultured on the medium containing different concentrations and combinations of growth regulators (NAA, BAP and Kinetin). Best response for shoot multiplication and elongation was obtained in medium with 0.75 mg/L NAA and 1.5 mg/L BAP. The individual shoots were transferred to half-strength MS medium supplemented with 0.5 to 2.0 mg/L of indole-3 acetic acid (IAA), indole-3 butyric acid (IBA) and naphthalene acetic acid (NAA) to observe root development. Half strength of MS + 1.00 mg/ L IAA has proven the best for root induction. The well rooted plants were hardened successfully in the potting mixture containing coconut husk, charcoal pieces (5-7 mm size), decaying litter and very small brick pieces (5-7 mm size) in the ratio of 1:1:1:1 and eventually plants are established under natural condition. The present investigation can be used for rapid mass propagation of this highly important Cymbidium finlaysonianum orchid species.

Micropropagation of an exotic ornamental plant, Calathea crotalifera, for production of high quality plantlets.

A successful protocol was established for micropropagation in two selected varieties of exotic ornamental plants, Calathea crotalifera. The effects of different sterilization techniques, explant type, and the combination and concentration of plant growth regulators on shoots induction were studied. The axillary shoot buds explants sprouted from rhizomes in soil free conditions showed high induction rate of shoots with lowest contamination percentage when treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl2. In the present study, the highest number of multiple shoots was obtained in MS basal medium supplemented with 3.5 mg/L 6-Benzylaminopurine (BAP), 1.0 mg/L 1-Naphthaleneacetic acid (NAA), 3% sucrose, and 6 g/L plant agar for both varieties and was used as multiplication medium. Microshoots were highly induced when the young shoot bud explants were incised longitudinally prior subculture. Chlorophyll analysis was studied to test the effects of activated charcoal and L-glutamine on reduction of necrosis problem. The maximum roots induction was recorded on MS medium supplemented with 1.0 mg/L 1-Naphthaleneacetic acid (NAA) compared to indolebutyric acid (IBA). The complete regenerated plantlets were successfully acclimatized in the soilless medium under greenhouse condition. This is the first report of rapid mass propagation for C. crotalifera.