Number Of Plantlets Research Articles

Efficient regeneration system for rapid multiplication of clean planting material of Ensete ventricosum (Welw.) Cheesman

Enset (Ensete ventricosum (Welw.) Cheesman) is an economically important staple food crop in Ethiopia, especially in the southern and southwestern regions. It is called “false banana” due to its resemblance to banana, but inability to produce any edible fruit. The crop is clonally propagated using field-grown suckers. This study reports the development of a robust regeneration technique to propagate large numbers of plantlets using corm discs containing intercalary meristematic tissues. Hundreds of shoot buds were induced from corm discs of enset cultivar ‘Bedadeti’ cultured on Murashige and Skoog (MS) medium supplemented with 1.5 mg L−1 2,4-dichlorophenoxyacetic acid, 0.216 mg L−1 zeatin, and 2 g L−1 activated charcoal. The shoot buds were regenerated into complete plantlets when transferred onto MS medium supplemented with 1 mg L−1 6-benzylaminopurine and 2 g L−1 activated charcoal. More than 100 plantlets were generated in 4 mo from corm discs isolated from a single in vitro mother plantlet. Well-rooted plantlets were acclimatized in soil with 100% success, and did not show any apparent phenotypic abnormalities under glasshouse conditions. This efficient regeneration system could be very useful for the rapid multiplication of clean pathogen-free planting material.

The Influence of Thidiazuron on Direct Somatic Embryo Formation from Various Types of Explant in Phalaenopsis amabilis (L.) Blume Orchid

Phalaenopsis amabilis is an important national flower of Indonesia as a parent for orchid breeding, so that needs a good strategy to produce high number of plants. The objective of this research is to analyze the use of thidiazuron (TDZ) for producing high number of plantlets, through directly induction of somatic embryos (SEs) from various explants. The method was used 20 each of protocorms, leaves, stems and roots as explants. The explants were dissected transversely, then put on various culture media: New Phalaenopsis (NP) and NP + (1, 2, 3) mgL−1 TDZ. Cultures were maintained at 25°C with continous white light. The formation of SEs was observed every week for 8 weeks. The results showed that SEs formation increased inline with the addition of TDZ concentration to the NP medium, for both velocity and amount of SEs formation. In NP0, SEs were formed at (26.07 ± 0.73) days after inoculation of protocorm, whereas on NP + (1, 2, and 3 mgL−1) TDZ, SEs were formed at (17.85 ± 0.67) days, (15 ± 0.64) days, and (11 ± 0.64) days, respectively. All types of explants formed SEs on NP + TDZ (1–3 mgL−1), whereas only 14 of 20 protocorms produced SEs (70%), and 8 of 20 stems formed SEs (40%) in NP0. In roots, SEs was formed on NP + 2 mgL−1 TDZ and NP + 3 mgL−1 TDZ. For stems, the highest amount of SEs (28.25 ± 1.07) was reached on NP + 3 mgL−1 TDZ, followed by protocorm (23.30 ± 1.13) SEs and roots (8.25 ± 0.68) SEs. In contrast, in NP0, the amount of SEs was very low (1.25 ± 0.46) from stem and (1.50 ± 0.65) from protocorms, there was no evidence of SEs formation in the leaves and roots.

Over-expression of KdSOC1 gene affected plantlet morphogenesis in Kalanchoe daigremontiana

Kalanchoe daigremontiana reproduces asexually by producing plantlets along the leaf margin. The aim of this study was to identify the function of the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 gene in Kalanchoe daigremontiana (KdSOC1) during plantlet morphogenesis. In this study, KdSOC1 gene expression was detected at stem cell niche during in vitro somatic embryogenesis and plantlet morphogenesis. Disrupting endogenous auxin transportation suppressed the KdSOC1 gene response. Knockdown of the KdSOC1 gene caused a defect in cotyledon formation during the early heart stage of somatic embryogenesis. Over-expression (OE) of the KdSOC1 gene resulted in asymmetric plantlet distribution, a reduced number of plantlets, thicker leaves, and thicker vascular fibers. Higher KdPIN1 gene expression and auxin content were found in OE plant compared to those of wild-type plant leaves, which indicated possible KdSOC1 gene role in affecting auxin distribution and accumulation. KdSOC1 gene OE in DR5-GUS Arabidopsis reporting lines resulted in an abnormal auxin response pattern during different stages of somatic embryogenesis. In summary, the KdSOC1 gene OE might alter auxin distribution and accumulation along leaf margin to initiate plantlet formation and distribution, which is crucial for plasticity during plantlet formation under various environmental conditions.

Sucker multiplication in plantain using chicken manure as a substrate supplement

This study was carried out to evaluate the response of chicken manure (CM) as a substrate supplement for sucker multiplication and its effect on varietal differences in two subgroups of plantain (Apantu, a False Horn and Apem, a French plantain), using well-developed sword suckers. The research was laid out in a 2 x 2 factorial randomized complete block design with two substrates (chicken manure/sawdust mix and sole sawdust substrate) and the two groups of plantain with three replications. Data were collected on temperature changes within the substrates, number of plantlets sprouted, physical growth of the plantlets, root formation of plantlets and their correlation with plantlet establishment in the nursery. It was observed that plantlet/substrate-types had no significant effect on number of plantlets sprouted. The corms of Apantu showed a higher positive response to all the treatments applied than those of Apem. Plantlets from CM/SD mix recorded better physical parameters but had lower survival rate as compared to the SD treatments. There was low positive correlation (26.79%) between rooted sprouts and survival rates of regenerated plantlets, which might be due to differences in environmental conditions. Generally, Apantu produced more vigorous sprouts than Apem, which may be due to differences in maturity period and varietal effect. Planting-material-type did not have any effect on plantlet acclimatization, while media-type showed greater impact on number of plantlets and acclimatization. CM/SD mix influenced higher sprouts but plantlets were poorly acclimatized to the environment. Key words: Chicken manure, sawdust, ‘Apantu’ (False horn), ‘Apem’ (French plantain), cultivar, media, plantlets.

In Vitro Culture of Ovules and Embryos from Seedless Grapes (Vitis vinifera L.)

Ovules of stenospermocarpic seedless grapes were cultured under different conditions. The number of embryos was not significantly increased when ovules of Muscat Seedless were cultured on a medium supplemented with the plant growth regulators indole-3-acetic acid and gibberellic acid compared to the basal medium. No correlation was found between the ovule size of Sultanina and the presence of embryos. Comparable percentages of embryos were found in small, medium and large ovules following weekly culture from four to 10 weeks after bloom. Varying results were obtained when ovules of four open-pollinated seedless cultivars were cultured on two basal media with or without activated charcoal. In all four cultivars the highest number of embryos was found at the later culture date. With three cultivars the best results were obtained when ovules were cultured on the medium of Bouquet & Davis (1989), supplemented with activated charcoal. Isolated embryos of Muscat Seedless and Sunred Seedless were cultured under various conditions and it was found that the absence of growth regulators resulted in the highest number of plantlets with normal leaves.

An Evaluation of Banana Macropropagation Techniques for Producing Pig Fodder in Northern Thailand

Smallholder farmers raising pigs in northern Thailand rely heavily on banana stalks as a fermented feed source, but struggle to reproduce banana plants fast enough to keep up with consumption. This study evaluated a variety of techniques for rapidly multiplying banana plants, using techniques appropriate and affordable to smallholder farmers in order to help meet this demand. Propagation techniques of Musa (ABB) cv. ‘Kluai Nam Wa’ were conducted in greenhouse and field experiments in both lowland and upland areas of Chiang Mai Province, Thailand. In greenhouse experiments, six treatments were conducted during the dry and rainy seasons, while five different treatments were compared in the field. Treatments used various methods of mechanical injury or application of benzyl aminopurine (BA) to induce plantlet differentiation. Number of plantlets to emerge, days to emergence, and circumference of plantlets were observed over a 90-day period. Results indicate that time of year plays an important role in the macropropagation of bananas, as significantly higher numbers of plantlets emerged during the rainy season. Plantlets emerged in 65 days, on average, during the dry season, but took only 54 days during the rainy season. During the rainy season, the presence of BA produced more plantlets than the other treatments, but during the dry season, there were no differences among treatments. Overall, the number of plantlets produced in all treatments evaluated was very low; however we believe this research is an important contribution to the literature and acknowledge that there exists significant opportunity to capitalize on the low-cost appropriate technology benefits that macropropagation of bananas can deliver to smallholder farmers.

Liquid medium culture method for rapid multiplication of banana (Musa acuminata) cv. ‘GRAND NAINE’ through tissue culture

For an increasing number of plantlets, liquid culture methods have demonstrated a number of important advantages over conventional semi-solid micropropagation, including several fold increase in multiplication rate, reduction in medium cost and also space, energy and lobour requirements. These cost-saving advantages have been the driving force for increased attention to the use of liquid systems. Effects of liquid medium using different simple low cost culture containers on in vitro propagation of banana cv. 'GRAND NAINE' were investigated and compared with conventional solid medium. The treatments studied included: Growtech container with nylon wire mesh support; simple polypropylene container with cotton fibre support; simple polypropylene container with partial immersion, simple polypropylene container with full immersion and conventional baby jar bottle with agar gelled medium. Simple polypropylene container with cotton fibre support was found effective than that of agar-gelled medium. The plantlets produced were also sturdier and better quality.

Rapid Development of PLBs Derived from Immature Seeds and Mass Multiplication of Vanda tessellata (Roxb.) Hook. Ex G. Don - A Threatened Orchid species in Bangladesh

A rapid and an efficient protocol for germination of immature seeds of Vanda tessellata, PLBs development and plant regeneration system have been developed. The results indicate that immature capsules performed better in terms of germination (100.0%) and development of PLBs (90.0%). Around 45 - 60 days old PLBs were transferred to half strength of MS supplemented with different concentrations of auxins and cytokinins. The highest weight of PLBs (5.53 g), the best plant regeneration (16.50) and shoot length (3.25 cm) were recorded when the medium was supplemented with BAP (1.0 mg/l) and NAA (1.5 mg/l). The highest number (5.0) and length (3.22 cm) of roots, the maximum number of plantlets (3.75) were obtained in the medium supplemented with 1.5 mg/l BAP and 1.0 mg/l IAA. The maximum numbers of leaves (3.33) were recorded in the medium fortified with 1.5 mg/l BAP + 1.00 mg/l IAA; the highest length of leaves (2.59 cm) was observed in the medium combining with of BAP and IAA (2.0 mg/l). Well shooted - and rooted plants were acclimated in pots that contained two types of compost. Here, type-II showed better performance on plant development.Plant Tissue Cult. & Biotech. 25(2): 181-191, 2015 (December)

The Application of Chitosan for Protocorm Like Bodies (PLB) Induction of Orchid (Dendrobium sp) In Vitro

Abstract Indonesia has a tropical orchid species diversity is greatest in the world, but unfortunately, the conservation of orchids less attention from the government. At present the development of orchids in Indonesia is still at the level of conventional farming, while conservation is still lacking. If no one focuses on the conservation of orchids in Indonesia will become extinct due to an increase in illegal logging, forest fires and climate change will further accelerate the extinction of orchids in Indonesia. While it's still a few who are able to do the development and utilization of special orchids by tissue culture technology in particular. The best method to date in the preservation and propagation of orchids is by tissue culture, because through tissue culture are many things that can be done in comparison with conventional methods. The effect of fungal chitosan on the growth and development of seed in culture was investigated solid medium. The growth of seed explants into Protocorm Like Bodies (PLB) in solid medium was accelerated up to 4 times in the presence of fungal chitosan. The aim of the research was induction PLB with difference concentration of chitosan were 5, 10, 15, 20 and 25 ppm. In general, the best result at 15 ppm of chitosan with number of PLB, fresh weigh of PLB and number of plantlet. This concentration will be used for mass production by bioreactor system.

In Vitro Callus Induction and Plant Regeneration from Stem Explants of Ceropegia noorjahaniae, a Critically Endangered Medicinal Herb.

An efficient protocol has been developed for in vitro regeneration of a large number of plantlets of Ceropegia noorjahaniae Ansari via indirect organogenesis from stem explants excised from in vitro-germinated seedlings. The callus was efficiently induced from the stem explants using Murashige and Skoog (MS) medium supplemented with auxins and their combinations. The highest number of shoots (16.0 ± 0.2) and shoot length (5.5 ± 0.1 cm) was achieved when the callus was subcultured to MS medium supplemented with 6-benzylaminopurine, BAP (2.0 mg/l) and indole-3-acetic acid, IAA (0.2 mg/l). The in vitro-developed shoots were rooted well in half-strength MS medium supplemented with 1.0 mg/l of indole-3-butyric acid (IBA) and 0.3 mg/l of α-naphthalene acetic acid (NAA). The plantlets were successfully hardened with 82 % survival rate. This is the first report on the regeneration of plants through indirect shoot organogenesis from stem derived calli of C. noorjahaniae.

Protocorm-like bodies and plant regeneration from foliar explants of Coelogyne flaccida, a horticulturally and medicinally important endangered orchid of Eastern Himalaya

An efficient induction of protocrom-like bodies (PLBs) and plantlet regeneration from the young leaves of in vitro grown seedlings of Coelogyne flaccida, an horticulturally and medicinally important endangered epiphytic orchid, was accomplished in order to develop mass-scale propagation. The young leaves (1.5 cm in length) from 110 days old aseptically germinated seedling were grown in vitro in Murashige and Skoog’s (MS) medium supplemented with different concentrations and combinations of NAA ( 0.5-2 mg/L), BAP (0.5-2 mg/L) and Kn ( 0.5-2 mg/L ). The explants produced protocorm-like bodies directly from the epidermal cells of leaf without the formation of intervening callus tissue within four weeks of culture. The highest number of plantlets regenerated through PLBs per explant after 15 weeks was 35-36 in presence of NAA (2mg/l) and Kn (2mg/l). Within 20-25 weeks individual plantlets produced 2-3 leaves and 2-3 roots. Chromosome number from all plants regenerated from leaf explants showed the same chromosome number as the mother plant as 2n = 40. During acclimatization, 80% of the plantlets survived after one month of transplantation.

Highly efficient protocol for callogenesis, somagenesis and regeneration of Indica rice plants

In the present study, we have reported a simple, fast and efficient regeneration protocol using mature embryos as explants, and discovered its effective applicability to a range of Indica rice genotypes. We have considered the response of six varieties in the steps of the regeneration procedure. The results showed that calli were variably developed from the scutellar region of seeds and visible within 6–20 days. The highest and lowest calli induction frequency (70% and 51.66%) and number of induced calli from seeds (14 and 10.33) were observed in MR269 and MRQ74, respectively. The maximum and minimum number (7.66 and 4) and frequency of embryogenic calli (38.33% and 20%) were recorded in MR219 and MRQ74, respectively. However, the highest browning rate was observed in MR84 (87%) and the lowest rate in MRQ50 (46%). The majority of plants regenerated from embryogenic calli were obtained from MRQ50 (54%) and the minimum number of plants from MR84. In this study, the maximum numbers of plantlets were regenerated from the varieties with highest rate of embryogenic calli. Also, various varieties, including MRQ50, MR269, MR276 and MR219, were satisfactorily responding, while MRQ74 and MR84 weakly responded to the procedure. Such a simple, successful and generalized method possesses the potential to become an important tool for crop improvement and functional studies of genes in rice as a model monocot plant.

Development of greenhouse soilless system for production of strawberry potted plantlets

The objective of this study was to produce high quality planting material in a soilless greenhouse system. Cv. Elsanta plants were planted in containers and set on a special rack in the greenhouse. Emerging plantlets were set (clipped with metal clips) in micro-pots filled with peat or coconut substrate and detached from mother plants after 7, 10 or 14 days. Efficiency of this nursery method depended on the number of runners emerging from mother plants and the number of plantlets on the runners. Plantlets rooted for a longer period showed greater tolerance to stress. Growing media used in the experiment did not have a significant influence on the dynamics and quality of plantlet rooting. Using the method developed in this study, all plantlets were properly rooted.

IN VITRO PROPAGATION OF NATIVE ORCHID DENDROBIUM SPECTABILE (BLUME) MIQ.

The forests of Papua and New Guinea have a rich variety of flowering plants. One orchid species from Papua and New Guinea is the Dendrobium spectabile. The D. spectabile has unique characteristics in which the flowers are slightly different in a florescent light. The flowers of D. spectabile are dissimilar with most of the other Dendrobium sp. Unlike the common Dendrobium, the D. spectabile will not flower without much higher levels of light. They will even take full sunlight for longer periods of time. The D. spectabile will start flowering when it reaches 4-5 years old. Recently, many people exploit this species, causing it to become very rare. Therefore, it is important to study the propagation method in order to conserve this species. In this study, in vitro propagation using different culture media were explored. The aim of this study is to compare two different media, i.e. medium VW with modification and medium W3, which has been known to give highest respond for growth of D. spectabile. The experiment was carried out at Tissue Culture Laboratory, CV Ayu Orchid Bali, from April-August 2012. The experiment employs Completely Randomized Design (CRD) design with 20 replications for each media. The result of the experiment showed that the highest respond for growth of D. spectabile was VW medium. This was indicated by higher number of plantlets (average 306.75), number of leaves (average 3.45) and plant height (average 2.65 cm). However, the leaves of plantlets using medium W3 showed dark green and the explants were stronger than explants in medium VW.

Improvement of Regeneration of Pelargonium radula via Somatic Embryogenesis

In vitro stem segments of Pelargonium radula cultured for callusing then differentiated into somatic embryos and subsequently regenerated plantlets. Initiation of callus was observed in culture medium containing low concentrations of the plant growth regulators, 2,4-dichlorophenoxyacetic acid (2,4-D) and/or α-naphthalene acetic acid (NAA). At 0.2 mg/L 2,4-D and 0.2 mg/L NAA was showing the highest rate (92%) of callus induction. The callus showed no sign of browning after sub-cultured. Sub-culturing the callus onto medium with 0.2 mg/L 2,4-D showed the highest in proliferation rate resulted 13.45g weight of callus. The presence of agar at 6 g/L and 0.5 mg/L Gibberellic acid (GA3) improved the regeneration of the somatic embryos, which produced maximum number of plantlets (15 plantlets). Agar with concentration of 9 to12 g/L increased the incidence of browning. The former medium was more successful in plantlet regeneration when the selected embryos were subsequently transferred to regeneration medium with 3 g/L agar, 0.5 mg/L GA3 and 0.5 mg/L Benzylaminopurine (BAP).

Direct Shoot Regeneration from Callus of <i>Melicope lunu-ankenda</i>

Melicope lunu-ankenda is commonly used in traditional medicine. The conventional propagation method for this species is inefficient due to low propagation rate and its lengthy period to maturity. In addition, insufficient planting materials often pose a problem for the plantation sector. The tissue culture technique is best alternative to overcome the problems. The callus induction and direct shoot regeneration protocols for M. lunu-ankenda were established. Callus was successfully initiated from leaves explants cultured in MS medium added with 2,4-D at concentrations 0.5 to 5.0 mg/L singly or in combination with NAA at concentrations 1.0 to 10 mg/L. Shoot was regenerated from callus in phytohormone-free medium, BAP at concentrations 0.5 - 5.0 mg/L singly or in combination of BAP with NAA or 2,4-D at concentration 0.5 and 1.0 mg/L, respectively. BAP at 1.0 mg/L induced the highest shoot regeneration rate (80%) and number of plantlet per calli. The established methods might be used for production of phytochemicals and plantlets in large scale.

Improved high-efficiency protocol for somatic embryogenesis in Peach Palm (Bactris gasipaes Kunth) using RITA® temporary immersion system

Bactris gasipaes Kunth (Arecaceae) is an Amazonian palm cultivated mainly for the production of fruits and heart of palm. Biotechnological tools based on somatic embryogenesis (SE) can be used for improvement and germplasm conservation of this species. The efficiency of different culture systems and culture medium composition were evaluated to optimize the efficiency of SE. The study also evaluated global DNA methylation levels, total protein and starch contents, and alcohol dehydrogenase (ADH) activity. The RITA® bioreactor showed enhanced multiplication of somatic embryos with increased protein, starch levels and ADH activity. Furthermore, a low DNA global methylation rate (27.52%) was observed, suggesting its relationship with the expression of proteins associated with the maturation of somatic embryos and their subsequent conversion to plantlets. Somatic embryo maturation was successfully achieved on Petri dishes and abscisic acid (ABA) supplementation in the culture medium. Chlorophyll pigmented somatic embryos were obtained in Petri dishes containing PGR-free culture medium, and RITA® provided the greatest number of plantlets to be acclimatized after reaching 6–7cm in length. In the acclimatization step, the best results were obtained with plantlets derived from RITA® after reaching 6–7cm in length. These new findings shed light on the importance of defining adequate culture systems for different steps of in vitro regenerative protocols and represent a breakthrough in the in vitro morphogenesis process of peach palm, resulting in a large number of somatic plantlets with enhanced physiological and morphological features.

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEML) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEML containing 0.5 μM picloram, 1 μM TDZ and 25 mg l−1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.

Enhancement of regeneration efficiency in banana (Musa acuminata cv. Berangan) by using proline and glutamine

Regeneration of Musa acuminata cv. Berangan (AAA) from embryogenic cell suspension was enhanced by using proline and glutamine. Sixteen embryo development media consisting of different concentrations of proline and glutamine were tested. Glutamine was preferred as the source of organic nitrogen supplement compared to proline. The embryos developed in liquid media were shown to produce 10.6-fold higher number of plantlets in lesser duration (1.5 to 2.5 months) as compared to solid media. Optimum somatic embryo development was observed in liquid media supplemented with 400 mg/l glutamine (M4GL) with an average regeneration of 33,844 ± 402 plantlets per ml of settled cell volume (SCV). Although both amino acids have proven to promote the embryo development, concentrations of proline between 200 to 400 mg/l in liquid media caused abnormal embryo differentiation.

Induction of Somatic Embryogenesis in Vulnerable Medicinal Plant Hedychium spicatum Buch-Ham ex Smith

This protocol has been developed for somatic embryogenesis in Hedychium spicatum. Simultaneously, a method has also been developed for the production of synthetic seeds by using somatic embryos. Direct somatic embryos were developed on cotyledon explants of zygotic embryos on MS supplemented with high concentration of NAA (20.0 µM). Induction of secondary embryogenesis was best in 2,4-D supplemented medium fortified with activated charcoal. Germination of somatic embryos was enhanced by using GA3. Besides this, round and semi-hard beads of somatic embryos (synthetic seeds) could be produced by using 2% Na-alginate and 100 mM calcium chloride and more than 30% germination of synthetic seeds was achieved in MS. Well acclimated plants produced via somatic embryogenesis and/or synthetic seeds were transferred to field where more than 60% survived. This simple study enabled us to obtain a number of plantlets throughout the year each cycle requiring a short period of time. Besides propagation, this study provided an ex situ method for conservation of this vulnerable Himalayan species.D. O. I.http://dx.doi.org/10.3329/ptcb.v23i2.17506Plant Tissue Cult. & Biotech. 23(2): 147-155, 2013 (December)