Plantlet Formation Research Articles

Optimization of an In Vitro Embryo Rescue Protocol for Breeding Seedless Table Grapes (Vitis vinifera L.) in Italy

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six “seedless × seedless” crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness.

Synthetic seed propagation of the therapeutic-honey plants

Context Increasing demand for therapeutic honey has driven establishment of Leptospermum nectar plantations. Methods for propagation involving synthetic seeds (artificially encapsulated miniature cuttings) may speed production of Leptospermum polygalifolium Salisb. and L. scoparium J.R.Forst. & G.Forst. Aims The study aimed to determine how nutrient strength of the encapsulation solution and the presence of benzyladenine (BA) in the emergence medium affect shoot and root emergence from synthetic seeds of L. polygalifolium and L. scoparium. Methods Nodes from in vitro shoots of three L. polygalifolium clones (P1, P6, P11) and two L. scoparium clones (S6, S12) were encapsulated in 3% sodium alginate with half- or full-strength Murashige and Skoog (MS) medium, and the synthetic seeds were placed on full-strength MS emergence medium containing 0 or 2.2 μM BA. Key results Full-strength MS in the encapsulation solution was effective for shoot emergence of both species. BA increased the percentage of synthetic seeds with shoot emergence in clone P6 but decreased the percentage in clone S12. BA stimulated shoot emergence through callus in clones P1, P6, S6 and S12, and increased the number of shoots per emergent synthetic seed in clones S6 and S12. Surprisingly, the simple use of full-strength MS medium without hormones was highly effective for adventitious rooting, stimulating root emergence and plantlet formation in 26–57% of L. polygalifolium and 100% of L. scoparium synthetic seeds. Conclusions These two Leptospermum species are highly amenable to propagation via synthetic seeds. A simple formulation of hormone-free, full-strength MS medium in the encapsulation solution and emergence medium provides high frequencies of plantlet conversion. Implications Synthetic seeds have potential to assist in mass production of Leptospermum plants for nectar plantations to meet demand for therapeutic honey.

ФАК ТОРЫ ПРЯМОГО ПРОРАСТАНИЯ МИКРОСПОРОГЕННЫХ ЭМБРИОИДОВ BRASSICA NAPUS L.

Isolated microspore culture is the main method of producing doubled rapeseed haploids and is widely used in research institutions and commercial companies. The protocol of rapeseed embryo production is well developed and efficient for many genotypes, but some issues remain due to the low regeneration frequency of plantlets from embryos. When the standard protocol is applied, regeneration of plantlets from microspore-derived embryos usually involves a callus-forming stage followed by regeneration of adventitious shoots or secondary embryos, which prolong the period of plantlet regeneration and makes production of doubled haploids complicated. Identifying factors, which affect the frequency of direct embryo germination will increase the frequency of plantlet formation and reduce the period of DH plant production. In this work, we studied the effect of medium pH on the duration of plantlet regeneration from rapeseed microspore-derived embryos and effect of their low temperature treatment of +1 and +5℃ for 3, 6, 8, 9, 12 days in complete darkness on the embryo maturation and germination. Rising the pH of the nutrient medium from 5.8 to 6.1 increased the frequency of direct embryos germination up to 18% and the overall frequency of plantlet regeneration up to 76%. Culturing embryos at low temperatures effected the frequency of direct germination of embryos into plantlets. The maximum frequency of 44–53% direct embryo germination was observed when cultured at +1℃ for 6 and 9 days, when embryos were cultured at +5℃ the frequency of direct germination was 0–10%. In the control variant without cold treatment it was 16%.

Promotion of Papua “Merbau” Iron Wood (Eusideroxylon Zwagerii) through in-Vitro Tissue Culture

Papua's ironwood comes from the merbau plant that inhabits the forests of Papua, one of which is in the Yapen Islands Regency, Serui City. In conventional cultivating ironwood for commercial purposes, there are various problems. When using powder and soil, a lot of sawdust is needed. In addition, the time required for planting seeds to produce shoots that are ready to be planted in the field takes a very long time of about 9-12 months. The first step in the cultivation of merbau tissue culture is to find the right formula for growth regulators. Auxins and cytokines are the right steps to stimulate merbau wood growth. It seems that the role of auxin (IAA) and cytokinin (BAP) in Murashige and Skoog media on Merbau Wood using cotyledon and meristem explants with good concentrations for shoot and plantlet formation was 1.0 mg/l IAA and 5, 0 mg/l from the latest research using hormones including NAA, Chitin and BAP in Merbau (Eusideroxylon zwageri) wood culture. NAA concentration 1 mg/l (A4B5) formed shoots and callus, A4B1 (NAA 1 mg/l; BAP 0 mg/l) resulted in organogenesis and NAA concentration 0.2 mg/l without cytokinins (A2B1) produced multiple shoots. The interaction of the combination of auxin NAA 1 mg/l with BAP 0.6 mg/l (A4B7) gave the best response in embryosomatic formation in implanted explants. While the interaction of NAA 0.2 mg/l with Kinetic 2 mg/l (A3B3) resulted in shoots and callus. The interaction of the combination of auxin NAA 1 mg/l with BAP 0.6 mg/l (A4B7) gave the best response in embryosomatic formation in implanted explants. While the interaction of NAA 0.2 mg/l with Kinetic 2 mg/l (A3B3) resulted in shoots and callus. The interaction of the combination of auxin NAA 1 mg/l with BAP 0.6 mg/l (A4B7) gave the best response in embryosomatic formation in implanted explants. While the interaction of NAA 0.2 mg/l with Kinetic 2 mg/l (A3B3) resulted in shoots and callus.

TARGET OF RAPAMYCIN is essential for asexual vegetative reproduction in Kalanchoë.

The unique mechanism by which leaf margin cells regain potency and then form a plantlet in Kalanchoë spp. remains elusive but involves organogenesis and embryogenesis in response to age, day length, nutrient availability, and drought stress. In light of this, we investigated whether TARGET OF RAPAMYCIN (TOR), a conserved protein kinase in eukaryotes that controls cell growth and metabolism in response to nutrient and energy availability, may regulate plantlet formation. Kalanchoë daigremontiana TOR (KdTOR) was expressed in the leaf margin at the site of plantlet initiation, in the early plantlet cotyledons, and in the root tip of the developed plantlet. Both chemical and genetic inhibition of TOR Kinase activity in Kalanchoë daigremontiana leaves disrupted plantlet formation. Furthermore, downregulation of KdTOR in transgenic plants led to wide-ranging transcriptional changes, including decreased K. daigremontiana SHOOTMERISTEMLESS and K. daigremontiana LEAFYCOTYLEDON1 expression, whereas auxin treatments induced KdTOR expression in the plantlet roots. These results suggest that the KdTOR pathway controls plantlet development in cooperation with auxin, organogenesis, and embryogenesis pathways. The ancient and highly conserved TOR Kinase therefore controls diverse and unique developmental pathways, such as asexual reproduction within the land plant lineage.

Occurrence of albinism during wheat androgenesis is correlated with repression of the key genes required for proper chloroplast biogenesis.

The phenomenon of albinism in wheat androgenesis is linked to the transcriptional repression of specific genes involved in chloroplast biogenesis during the first weeks of in vitro culture. Isolated microspore culture is widely used to accelerate breeding programs and produce new cultivars. However, in cereals and particularly in wheat, the use of this technique is limited due to the high proportion of regenerated albino plantlets. The causes and mechanisms leading to the formation of albino plantlets in wheat remain largely unknown and, to date, no concrete solution has been found to make it possible to overcome this barrier. We performed a molecular study of proplastid-to-chloroplast differentiation within wheat microspore cultures by analyzing the expression of 20 genes specifically involved in chloroplast biogenesis. Their expression levels were compared between two wheat genotypes that exhibit differential capacities to regenerate green plantlets, i.e., Pavon and Paledor, which produce high and low rates of green plants, respectively. We observed that chloroplast biogenesis within wheat microspores was affected as of the very early stages of the androgenesis process. A successful transition from a NEP- to a PEP-dependent transcription during early plastid development was found to be strongly correlated with the formation of green plantlets, while failure of this transition was strongly correlated with the regeneration of albino plantlets. The very low expression of plastid-encoded 16S and 23S rRNAs within plastids of the recalcitrant genotype Paledor suggests a low translation activity in albino plastids. Furthermore, a delay in the activation of the transcription of nuclear encoded key genes like GLK1 related to chloroplast biogenesis was observed in multicellular structures and pro-embryos of the genotype Paledor. These data help to understand the phenomenon of albinism in wheat androgenesis, which appears to be linked to the transcriptional activation of specific genes involved in the initial steps of chloroplast biogenesis that occurs between days 7 and 21 of in vitro culture.

In vitro propagation and phyto-chemical assessment of Cymbidium aloifolium (L.) Sw.: An orchid of pharma-horticultural importance

Orchids are one of the most fascinating plant families amongst the flowering plants. Amongst the various orchid genus Cymbidiums figure out prominently. The present study focuses on the development of an efficient in vitro regeneration protocol for Cymbidium aloifolium, an epiphytic orchid with high commercial value. It also aims at analysing the important phytochemicals and antioxidant potential of in vitro and in vivo plants. The effects of MS basal media (half or full strength) and MS medium supplemented with BAP were studied for in vitro seed germination. Among different media, 1/2 MS medium resulted in in vitro seed germination, protocorm development and plantlets formation in the shortest duration. However, 6-Benzylaminopurine (BAP) played an important role during proliferation of protocorms or in vitro multiplication, thus resulted in maximum number of shoots (39), root length (6.10 cm) on MS medium with 1.0 mg/L BAP. Largest shoot length was observed on solid BM-2 medium. 90% of the plantlets survived in the potting mixture consisting of cocopeat, perlite and wood saw dust in a ratio of 1:1:1. Comparative phytochemical analysis revealed that in vitro regenerated plants showed a higher yield of secondary metabolites. This work will facilitate the utilization of orchid phyto-constituents for various pharmaceutical applications in the industries.

TISSUE CULTURE MULTIPLICATION OF Paphiopedilum insigne DEPENDING ON THE MEDIUM TYPE, GROWTH REGULATORS AND NATURAL SUPPLEMENTS

Paphiopedilum is an ornamental orchid used mainly for interior decoration. As the division of plants is uneconomical, a fast method of propagation is needed. The aim of this study was to evaluate the influence of medium type (MS or VW), growth regulators, i.e. BA, KIN, TDZ used separately or in combinations and natural additives, i.e. coconut water, banana pulp, casein hydrolysate, on Paphiopedilum insigne plantlets grown in vitro. It was found out that BA in concentration of 0.5 mg·dm–3 allowed to obtain the highest multiplication rate (2.92), however the use of KIN in concentration of 1 mg·dm–3 resulted in formation of bigger and higher quality plantlets, It is possible to replace cytokinins with other biologically active substance, such as banana pulp. The 1/2 MS medium might be used for Paphiopedilum insigne tissue culture, as there was no difference in terms of multiplication rate and the obtained plantlets were of better quality, especially that it is cheaper and easier to prepare.

Effect of Plant Growth Regulators on Somatic Embryogenesis and Plantlet Development of Turkey Berry (Solanum torvum SW)

In the present study it was reported on direct somatic embryogenesis and plant regeneration from cotyledon and leaf explants of Turkey berry/pea egg plant (Solanum torvum SW), a medicinally important plant. Somatic embryogenesis has several advantages over other routes of in vitro plant regeneration. Somatic embryogenesis was induced directly from cotyledon and leaf explants on MS medium fortified with BAP (0.5 mg/L)+NAA (0.5-6.0 mg/L). High percentage of somatic embryogenesis (90%), maximum number of somatic embryos formation (62±0.18) along with high percentage (76%) conversion of somatic embryos into bipolar embryos was observed on cotyledon explants in 0.5 mg/L BAP+2.5 mg/L NAA. At the same concentration of BAP (0.5 mg/L)+NAA (2.5 mg/L) also resulted on the maximum percentage of somatic embryogenesis (92%), the highest number of somatic embryos formation (88±0.15) and the highest percentage (76%) of somatic embryos conversion into bipolar embryos in leaf explants. A mixture of globular, heart and torpedo-shaped embryos were germinated on MS medium supplemented with 0.5 mg/L IAA+1.0-4.0 mg/L BAP. Maximum germination frequency (75±0.14) of somatic embryos and plantlet formation was found in 0.5 mg/L IAA+2.0 mg/L BAP, but they didn’t germinate on ½ MSO and MSO media. The survival rate of regenerated plants after field transfer was recorded to be 75%. These regenerated plants were found morphologically similar to donor plants. The present protocol can be used for conservation of the species and also for genetic transformation experiments in S. torvum.

Nitric oxide mitigates thalidomide-induced abnormalities during germination and development of fennel seeds

Thalidomide causes teratogenic effects in several animal species and in humans. Accordingly, the World Health Organization banned thalidomide when mothers who took thalidomide during pregnancy delivered abnormal fetuses. After four decades, thalidomide underwent drug "re-purposing" since its antiangiogenic and immunomodulatory effects were therapeutic for multiple myeloma. There are no reports of thalidomide's effects on prokaryotes, but it showed teratogenic effects in Arabidopsis thaliana, an ancestor of the plant kingdom. This proof of concept study clearly shows that thalidomide caused a significant and reproducible decrease in germination rate, nitric oxide (NO) production, and chlorophyll content of fennel plantlets. Thalidomide also induced the formation of abnormal fennel plantlets with stunting, wrinkling, and curling of fennel shoots and leaves. Notably, quantitative analysis showed that thalidomide caused a 50% increase in the formation of abnormal fennel plantlets and that these negative effects of thalidomide showed a 2.50- to 4-fold decrease when fennel seeds were co-incubated with an NO donor (Spermine NoNoate) or a stable cGMP analog 8-bromo Guanosine 3',5'-cyclic monophosphate (8-Bromo-cGMP). This study is important because it confirms that thalidomide's negative effects on fennel seed germination and growth are mediated by attenuation of NO and disruption of NO signaling. This reproducible model of thalidomide-induced, NO-dependent damage in a plant system can be used to further investigate the molecular mechanisms of thalidomide action in plants. Importantly, this study establishes a link between the evolution of development of higher plants and mammals.

Banana Cultivars Microshoot Induction and Plantlet Formation Using Cytokinin and Auxin

<p>Banana is a horticultural plant with very high potentials, which contains carbohydrates and vitamins that are useful in fulfilling people's food and nutritional needs. Hence, this study aims to produce superior banana seedlings and develop a protocol for their mass production using a plant <em>in vitro</em> culture technique. This was a two stage-experiment i.e. microshoot production and plantlet formation. The result showed that Gebyar cultivar produced more shoots than the Kepok Kuning<em> </em>cultivar, with an average of 4.25 microshoots explant<sup>-1</sup>. However, Kepok Kuning produced more leaves than Gebyar,<em> </em>with an average of 4.64 leaves plantlet<sup>-1</sup>. Banana shoots cultured on the media containing Indole-3-acetic acid (IAA) at a concentration of 2.5 µM produced the highest leaves number. Meanwhile, those cultured on the media containing 1-Naphthalenesacetic acid (NAA) at a concentration of 7.5 µM produced the highest roots number. A Murashige and Skoog (MS) medium supplemented with 6-Benzylaminopurine (BAP) up to 30 µM and the one supplemented with 7.5 μM of NAA are suitable for Kepok Kuning and Gebyar<em> </em>cultivars micropropagation with regard to microshoot induction and plantlet formation, respectively.</p>

In vitro induction of oil palm (Elaeis guineensis Jacq.) shoot roots and their acclimatization in mycorrhiza-enriched media

Oil palm is a vegetable oil-producing plant (CPO) which provides the largest foreign exchange contribution compared to other crops and is widely used in food, medicine, cosmetic, and energy industries. Tissue culture technology is currently used to produce quality oil palm seeds. Oil palm shoots tend to grow and develop in clumps (groups) in vitro. Bipolar nature does not appear in all the shoots produced, so to produce plantlets it is necessary to do induction. This research aimed to obtain the right root induction media. A completely randomized design (CRD) with two factors was used with the first factor being the type of auxin (IAA, IBA, and NAA), and the second was the auxin concentration (0, 0.25, 0.5, and 0.75 ppm). In the eighth week after planting, the variables of root length, number of leaves, and shoot height were not significantly different except for the root number. The best root induction media for plantlet formation was the MS base medium with the addition of NAA type auxin at a concentration of 0.75 ppm. The plantlets formed a symbiosis with mycorrhiza which was applied at a dose of 4 g per polybag in the fourth month after planting.

Embryo Culture of New Hybrid of Pecan (Carya Illinoensis) with Walnut (J. Regia and J. Nigra)

Present study was conducted to achieve intergeneric hybridization in Juglandaceae for the first time. Due to embryo abortion possibility, the embryo rescue technique was conducted via callus induction from immature hybrid embryo culture. For this reason, embryos were sampled at 20, 30, 40 and 60 days after pollination and cultured on two enrichment media (WPM and DKW). The developmental stage of immature embryo, kind of medium and addition of growth regulators to the induction medium played the role in promoting better callus and plantlet formation. The best quality and quantity of callus induction were achieved in 60 days’ explant. Maximum induction was in WPM and there was no callus induction in PGRs free medium. Browning was the most obstacle to conquer in callus proliferation. Germination of remained mature hybrid embryo until fruit harvest time was induced on three media (MS, WPM, and DKW). There were no significant differences between medium and male parents effect on germination parameters. But, MS and WPM performed better for hybrid embryo germination. The first germination was 7 days in MS and WPM and about 10 days for DKW after culture. In all medium the roots were robust and more developed than the leaves. The microplant transferred to soil after hardening.

Hybridization and In Vitro Seed Germination of a Commercial Hybrid Oncidium Orchid in Indonesia

Oncidium is the one of orchid family that is not native Indonesia. In the development of Oncidium breeding are widely encountered difficulties, especially when using advanced commercially hybrid varieties. Hybridization and seed germination of Oncidium is still not established yet. The objectives of this study were to obtain population of hybridization and to identify the best medium culture of seed germination and planlet development of a commercial Oncidium Orchid in Indonesia. The observations were made at a percentage of the success of crosses, seed maturity, the length of the protocorm formation, the effect of media type and sucrose concentration. In this study, the percentage of the cross-success of Oncidium was 15.7% (85 capsules) from 566 crossing and 6.18% (35 capsules) germinated. The fruit harvest age of Oncidium crosses varies between 165 days to 245 days after crossing. The duration of protocorm germination varies between 17-82 days after spreading seed. Protocorms were growth well and fast on medium Tsuchiya supplemented with 1 mg L−1 BAP and 0.25 mg L−1 NAA. and plantlet formation were promoted on the same based media Tsuchiya but supplemented using 0.5 mgL−1 BAP and 0.1 mg L−1 NAA. The optimized protocol required about 24–30 weeks from the spread seed to the plantlet formation. Seedling were grown at size 5-7 cm immediately planted as pot community. The time of acclimatization varies between 6-8 months after the last subculture. while individual time ranges between 4-5 months after acclimatization. Hopefully, the current study will assist with future development of Oncidium Orchid breeding in Indonesia.

Indirect organogenesis and somatic embryogenesis for regeneration of Rauvolfia serpentina L. from root explants

Highest frequencies (75%) of friable embryogenic nodular calli were recorded from the root explants in the presence of MS + 4.0 mg/l BAP and 4.0 mg/l NAA. In combinations of BAP and NAA 45.4% callus produced direct plantlets formation and 36.3% only from aerial part of shoots. MS medium with 1.0 mg/l BAP and 0.5 mg/l Kn produced compact callus where 13.3% produced direct plantlets formation and 45.7% produced only shoot formation. Well-developed globular somatic embryos were found to form when the callus were cultured more than 6 - 8 weeks on MS with 1.0 mg/l BAP and 0.5 mg/l Kn. After acclimatization the well rooted plantlets were transferred to pot and grown successfully in soil.

Plantlet Formation and Acclimatization of Sugarcane cv. PS 881 with Different Types and Concentration of Auxin

This research has been carried out with a view to induce rooting and plantlet formation, followed by acclimatization. Micro shoots of sugarcane cv. PS 881 were cultured on Murashige and Skoog medium supplemented with different types and concentration of auxins for root induction. This research conducted experimentally using a split-plot design. The main plots were three types of auxins, which consisted of IAA, IBA, and NAA. The subplots were auxin concentrations with four levels, i.e. 0 I¼M, 5 I¼M, 10 I¼M, and 15 I¼M. Significantly faster root emergence time and higher number of roots observed in the Murashige and Skoog basal medium supplemented with 10 I¼M NAA. The best root length obtained in the Murashige and Skoog basal medium supplemented with NAA 0 I¼M. Plantlets derived from NAA 10 I¼M treatment showed the best performance during acclimation with a 100% survival rate. NAA at a concentration of 10 µM considered to be the best treatment in plantlet formation and acclimatization of sugarcane cv. PS 881. This study showed that the use of MS medium with 10 uM NAA is able to increase the growth of PS 881 sugarcane plantlets. The results of this study can increase the availability of high quality seedlings and increase national sugar production.

Effect of Environmental Factors on Successful in vitro Morphogenesis of Madhuca longifolia

Madhuca longifolia is a commercially important tree species commonly known as mahua. The livelihood of large populations of tribal people depends on collection of its flowers and seeds. Almost all the parts of Mahua are utilized in diversified uses like in industry as artificial sweetner, biodiesel, food products, in soap industry etc. In the present study, a successful attempt was made to establish in vitro cultures of Mahua from nodal segments and factors influencing in vitro morphogenesis were evaluated as propagation through seeds and cuttings encounters problems. Axillary bud break (64.44%) was successfully achieved by culturing nodal segments on Murashige and Skoogs (MS) medium supplemented with 3 mg l-1Benzyladenine (BA) in nodal explants collected during the months ofJuly-September (rainy season).Shoot multiplication with maximum number of shoots, maximum number of leaves and longest shoots was achieved on MS medium supplemented with 3 mg l-1 BA when a subculture cycle of 30 days was followed. On MS medium supplemented with 2 mg l-1 Indole-3-Butyric Acid (IBA), in vitro excised shoots were successfully rooted (55.55%) after 40 days. A two step method was employed for successful hardening of rooted plantlets. Firstly, the plantlets were transferred for one week in 1/2 strength of MS liquid medium. Then, the plantlets were transferred to root trainers containing soilrite soaked with inorganic salts of ½ strength MS medium. The hardened plantlets were acclimatized firstly in a mist chamber and then in polybags in shade house. The present study provides an effective means for in vitro shoot regeneration and plantlet formation through nodal segments of Madhuca longifolia, a commercially important tropical tree with multifarious uses.

Increasing the somatic embryogenesis frequency of Panax vietnamensis Ha et Grushv. by disinfection of leaf explant using nano silver and the addition of nano silver in culture medium

Somatic embryo is a developmental method for mass multiplication of valuable medicinal plants. In this study, leaf explants of Ngoc Linh ginseng were disinfected with nano silver at different concentrations and exposure times to eliminate infectious agents and induce embryogenic callus for the production of somatic embryos. The results show that the lowest contamination rate (20.00%) was observed when leaf explants were treated with 0.5 g/L nano silver for 15 minutes while the highest embryogenic callus induction rate (72.22%) and fresh weight (0.77 g) was determined at 0.2 g/L nano silver for 20 minutes. High frequency of somatic embryogenesis formation and germination were occurred on MS medium supplemented 1.0 mg/L 2,4-D; 0.5 mg/L NAA; 0.2 mg/L Kin and 1.6 mg/L nanosilver. After 8 weeks of culture, the number somatic embryos derived from nano silver treated-leaves was increased 2 times than non-treated explants. Addition of 1.0 mg/L NAA and 1.2 mg/L nano silver was showed the highest shoot and root length, root number, fresh and dry weight of plantlets. This research showed that pre-treatment and supplement of nano silver in culture medium is potentially useful for improving embryogenesis frequency, and plantlet formation of Ngoc Linh ginseng cultured in vitro.

Propagasi in vitro tanaman kurma (Phoenix dactylifera L.) pada bioreaktor dengan perendaman sesaat

The cultivation of date palm in Indonesia has increased since the last decade. However, the superior date palm seedlings are still limited and most of them are imported from other countries. The mass supply of superior date palm seedlings can be provided by in vitro propagation in the bioreactor. Therefore, the research was conducted to develop a protocol of date palm in vitro propagation by using Temporary Immersion Bioreactor (TIB). The in vitro propagation was carried out through somatic embryogenesis technique using meristematic tissues isolated from offshoots of date palm female clone cv. Zambli as explants. The explants were sterilized and then cultured to produce embryogenic calli and somatic embryos. Afterwards, somatic embryos germination and plantlets formation were conducted in TIB with treatments of immersion period: 3, 10, and 30 minutes every 6 hours, with 8 replications, The results showed that the optimal somatic embryo germination in TIB was with the immersion period of 30 min every 6 h, resulting in the most formation of shoots and fresh biomass weight increment up to nearly threefold in 6 weeks. Thereafter, plantlets formation in TIB with immersion period of 10 min and 30 min every 6 h exhibited similar performances in producing more plantlets with higher total fresh weight and better vigor than those of 3 min every 6 h. However, there were more rooted plantlets in the TIB with immersion period of 10 min every 6 h. Based on the results, an in vitro propagation protocol via somatic embryogenesis in TIB has been successfully developed for mass propagation of date palm cv. Zambli, which produced plantlets with good vigor and rooting.

DO JASMONIC ACID AND ACTIVATED CHARCOAL INCREASE THE in vitro DEVELOPMENT OF ORANGE CARROT (Daucus carota L.) AND PURPLE CARROT (Daucus carota L. ssp. sativus var. atrorubens Alef.)?

Present study aimed to reveal the effects of jasmonic acid and activated charcoal on in vitro carrot plantlet regeneration by using Murashige and Skoog (MS) medium supplemented with BAP, NAA, activated charcoal and jasmonic acid at various concentrations. To serve the purpose, in vitro carrot seed germination, shoots, cotyledons, and first leaves formation of orange and purple carrot plantlets were investigated. During the experiments, root size, weight, and size of petiole diameter, hyperhydricity and callus formation rate were recorded. Experimental results revealed that combination of jasmonic acid and activated charcoal in medium had a positive effect especially on the first stage of developmental processes such as seed swelling and germination, cotyledon and first leaf formation as well as having positive effects on above-ground internode elongation, petiole and plantlet height.