Tissue Culture Laboratory Research Articles

Peranan BAP dan Air Kelapa pada Medium VW terhadap Organogenesis Dendrobium sp.

Dendrobium orchids are a favorite genus for orchid lovers than other orchids, because of their ability to easily adapt to various environments. The production of orchids in Indonesia is very low, while the demand is increasing from year to year, so it needs to be accelerated with tissue culture in vitro. This in vitro using VW media as a determing factor in plant propagation, VW media is a medium that is often used for plant propagation. The purpose of this research was to determine the function of BAP and coconut water on organogenesis of orchid explants of Dendrobium sp. This research is done at the Tissue Culture Laboratory FMIPA, University of Islam Malang, started from September to November 2020. This study used an experimental method and a Completely Randomized Design (CRD). This research consisting of 2 treatments with 3 replications each, BAP 0,5 ml, 1 ml and, 1,5 ml, and coconut water 100 ml, 150 ml and 200 ml. The results showed that the organogenesis the amount of shoots, amount of leaves, the highest amount of roots and root length in the BAP treatment with a concentration of 1,5 ml, with an average of 1.3 shoots, 3.86 leaves, 0.86 roots and root length 0.4 centimeters. While the coconut water treatment showed the highest organogenesis the amount of shoots, amount of leaves, the highest amount of roots and the root length with a concentration of 150 ml, with an average of 0.63 shoots, 3.53 leaves, 1 root and root length 1.66 centimeters. Coconut water treatment more effective in root extension with an average of roots 1 and 1,66 centimeters of root length. While BAP treatment, more effective in shoot development with an average of shoots 1,3 and the average of leaves 3,86.
 Keywords: BAP, Organic material, Dendrobium, Organogenesis.

Farklı Besi Ortamlarının Tatlı Patateste in Vitro Çoğaltım Üzerine Etkil

This study was carried out in Tokat Gaziosmanpaşa University, Faculty of Agriculture, Field Crops Department, Industrial Plants Tissue Culture Laboratory in 2020. In the study, effects of different media (LS media, LS+1 mg/L Gibberellic acid (GA3), 0.1 mg/L kinetin (KIN) and LS+0.5 mg/L indole-3-acetic acid (IAA) on sweet potato genotypes (Havuc and Hatay Yerlisi) were investigated. Plant and root weights, plant and root lengths, and number of nodes and roots were determined. In this study, plants grown in cytokinin and gibberellin media had higher root number, root weight, root length, plant height, number of nodes and plant weight compared to the ones grown in auxin media. Havuc local genotype had higher values in terms of the investigated traits in all of the media studied.

Goat’s Milk as a Potential Anti-proliferative against Colon Cancer Cell Lines

Aim: To investigate anti-proliferative effect of fresh and pasteurized goat milk against colon cancer cell lines (HT-29, HCT-116, CT-26).
 Study Design: Experimental study.
 Place and Duration of Study: Central Laboratory, Tissue Culture Laboratory, Universiti Sultan Zainal Abidin, Terengganu between January 2020 and April 2020.
 Methodology: Samples comprised of two types goat milk, which were fresh and pasteurized in powder form. The samples were analysed for the anti-proliferative effect by MTT assay, and IC50 value was determined. Then, cell apoptotic changes were observed by light inverted microscope by 24, 48 and 72 hours.
 Results: Experimental data showed that the fresh sample produce the highest yield (9.40%) than the pasteurized sample (7.17%). The fresh sample yielded the most potent cytotoxic value (0.28 ± 0.03), followed by pasteurized sample with value IC50 0.32 ± 0.02 against HCT-116 cells. Then, the anti-proliferative effect was observed on cell apoptotic changes by reduction of cell volume, cell densed, and presence of fragmentation and apoptotic bodies at 24, 48 and 72 hours treatment.
 Conclusion: In conclusion, the fresh sample of goat milk yielded the potent anti-proliferative effect than pasteurized sample.

​Effect of Different Media on In vitro Rooting in Different Cultivar of Lilium longiflorum Cv. Elite, Brunello, Cordelia

Background: Lilium (Lilium longiflorum Thunb.) belongs to the family Liliaceae and is a native of Northern Hemisphere (up to South Canada and Siberia). Conventionally Lilium can easily be propagated by sexual and asexual methods of propagation but these prevalent methods are not capable of meeting the increasing demand in domestic and global market. Generally, Lilium is propagated through bulbs but, limited number of bulbs per plant, long dormancy period of bulbs which again results into non-availability of planting material throughout the year. Keeping in view the above facts, the present study was undertaken with the following objective: “To standardize the cost effective protocol for micro propagation of lilium to produce disease free and true to type plants at a faster rate”. Methods: The present investigation was carried out in the Tissue Culture Laboratory of the Centre for Research and Application in Plant Tissue Culture. The experiment was laid out in a C.R.D. (Factorial) with three replications. In vitro raised bulblets were separated out and were transferred on to the root regeneration media. Different levels of NAA were used in MS media for the rooting of in vitro raised bulblets and percent rooting of plantlet is recorded. Result: It was interesting to note that the media LR-3 (MS + NAA 1.0 mg/l) is most efficient for rooting in all type of cultivars. All the three cultivars used responded very poor on media LR-1 (MS basal).

Plant Tissue Culture Research and Development in Ethiopia: A Case Study on Current Status, Opportunities, and Challenges

Plant tissue culture (PTC) is one of the well-adapted and practiced biotechnology tools in Ethiopia. After the initial success of tissue culture at the Ethiopian Institute of Agriculture Research and Addis Ababa University, the technology spread its roots to most of the research institutes and universities across the country. Consequently, the technology continues to extend at the commercial level. However, there is a lack of information at the national level on the capacity of national institutes in terms of available infrastructure and skilled human power, the potential of the laboratories for tissue culture-related activities, and existing challenges that hinder tissue culture research and development. This study aimed to assess the status of tissue culture research and development in Ethiopia and identify gaps that hinder its application in various economic sectors. The primary data were collected from 19 tissue culture laboratories across the country using questionnaires and physical observation. Accordingly, the results of the study revealed that Jimma Agricultural Research Center from the federal research institute and Amhara Regional Agriculture Research Institute, Bahir Dar Laboratory, from the regional research institute are relatively well equipped with facilities and equipment for tissue culture R&D activities. Among the commercial laboratories, Tigray Biotechnology Center is well equipped and has the highest production capacity with 40 million seedlings per year. Besides, the assessment revealed that a total of 54 plant species have been under in vitro protocol optimization and development process across all PTC laboratories. Nevertheless, tissue culture application in public and private sectors in Ethiopia is limited by several technical, administrative, and financial constraints. Therefore, to exploit the maximum benefit of PTC technology in the existing facilities, further intervention is critical in solving the identified challenges.

Pengaruh Pemberian GA3 terhadap Perkecambahan Embrio Somatik Kakao

<p><em>Germination process of somatic embryos is one of critical steps in somatic embryogenesis. The conversion of somatic embryos into plantlets is still low and adding kinetin, ABA, NAA, and GA<sub>3</sub> to the germination medium of cacao somatic embryos may increase it. The study aimed to investigate the effect of GA<sub>3</sub> on somatic embryo germination at the cotyledonary phase. The study was conducted in the Indonesian Industrial and Beverage Crops Research Institute (IIBCRI) Tissue Culture Laboratory from May to November 2017. The study used a factorial completely randomized design (CRD) with seven replications. The first factor was the cacao genotypes, namely SCA 6 and MCC 02, and the second factor was four concentrations of GA<sub>3</sub>, namely 0.0; 1.0; 3.0; and 5.0 mg/l. Characters observed were the percentage of shooting cotyledonary embryos, the percentage of rooting cotyledonary embryos, the percentage of shooting and rooting cotyledonary embryos, percentage of browning cotyledonary embryos, root length, shoot length, and hypocotyl length at one and two months old. The results showed no interaction between genotypes and GA<sub>3</sub> concentrations on all observed characters. Adding GA<sub>3</sub> at 1,0 mg/l concentration tends to increase the percentage of shooting cotyledonary embryos of SCA 6 and rooting cotyledonary embryos of MCC 02. SCA 6 clone has a higher percentage of shooting cotyledone, shooting and rooting cotyledone, and browning cotyledone compared to those of MCC 02.</em></p>

Screening of Salt Tolerant Potato Genotypes using Salt Stress and Molecular Markers

Aims: To screen potato genotypes for salt tolerance using in vitro salt stress and moleulcar markers.
 Experimental Design: The experiment was arranged in Completely Randomized Design (CRD) with three replications.
 Place of the Study: The experiment was conducted at the Molecular Biology and Tissue Culture Laboratories, Institute of Biotechnology and Genetic Engineering, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, Bangladesh.
 Methodology: In vitro screening of five potato genotypes (CIP 112, CIP 117, CIP 120, CIP 127, and CIP 128) was done in an agar medium using tissue culture technique at different concentrations of salt viz. 0.0, 10, 20, 40, 60, 80, 100, 120 mM of NaCl. All genotypes were further analysed through SSR markers using three primers. The DNA bands were visualized on gel electrophoresis and scored for polymorphic loci, gene diversity, and genetic distance. NTSYSpc program was used for constructing unrooted neighbor-joining tree and scatter diagram.
 Results: Potato genotypes CIP 112 and CIP 117 emerged as the most salt tolerant genotypes with the highest plant height, shoot dry weight, root length, and root dry weight at different concentrations of NaCl. CIP 127 and CIP 128 were poorly tolerant to salt stress. The most sensitive genotype CIP 120 produced minimum plant height, shoot dry weight, root length, and root dry weight at different NaCl concentrations. Results indicated that significant differences were found among cultivars. The banding pattern of microsatellite confirmed a distinct polymorphism among salt tolerant, moderately salt tolerant and salt sensitive lines. The clustering pattern of the potato genotypes suggests that variations occur due to genotypic variation and possibly not epigenetic adaptation under salt stress conditions.
 Conclusion: The salt tolerant pototo genotypes CIP 112 and CIP 117 can be used for developing salt tolerant genotypes.

The effect of explant size and addition of antiviral ribavirin on proliferation of meristematic potatoes (Solanum tuberosum L.)

Potato (Solanum tuberosum L.) belongs to Solanacearum genus which is vegetatively propagated through tubers or cuttings. Viral systemic diseases are an important disease that can be carried within seeds. Conventional techniques of tissue culture when combined with chemotherapy can eliminate systemic diseases, especially viruses. Research activity was carried out in the tissue culture laboratory of Indonesian Vegetable Research Institute (IVEGRI) from April until November 2018. The study was aimed to observe the effect of (1) antiviral Ribavirin concentrations (0; 5; 10 mg l−1) added into MS medium supplemented with GA3 0.15 mg l−1, kinetin 0.1 mg l−1, Calsium panthothenate 2 mg l−1, Myo inositol 100 mg l−1, coconut water 100 ml l−1, sucrose 30 g l−1, agar 6.5 g l−1, pH 5.7; and (2) genotypic difference of given varieties i.e. Granola, Median and Atlantic to the growth and development of the meristematic tissue (E1), shoot tip (E2). The results showed that growth percentage and development of meristematic shoot at 4 weeks after treatment was 50 – 80%, contamination rate was 20 – 50%; the percentage of normal growth was 40 – 50%, average number of shoots was 0.25 – 0.95, the number of nodes was 0.35 – 2.50, roots 0.20 – 0.93; while visual observation of antiviral Ribavirin on MS medium showed influences on growth where explants of shoot tip growth was better than meristem of the three varieties studied.

Effect of Silver Nanoparticles, Medium Composition and Growth Regulators on in vitro propagation of Picual Olive Cultivar

Micro-propagation of olive provides the possibility of producing clones of native plants with appropriate attributes and mass production in short period of time, compared to conventional methods. Micropropagation of olive requires optimization of protocols and adaptation to specific cultivars requirements. This experiment was carried out during 2020 and 2021 seasons in tissue culture laboratory of Pomology Department, Faculty of Agriculture, Cairo University in order to evaluate the potential effects of nano silver at (0, 5, 10 and 20 mg L-1) to improve sprouting percentage and shoot growth of Picual olive cv. The effect of nutrient media composition (MS modified, OM and OM modified) and cytokinins (BAP and Zeatin) on multiplication rate and shoot growth were examined. IBA and NAA (2 and 4 mg L) was examined during rooting stage. The obtained results cleared that Silver Nanoparticles (AgNPs) at 5 mg L-1 recorded the highest percentage of bud sprouting, shoot length, number of shoot/explant and number of leaves/ shoot. Olive medium (OM) recorded the highest shoot length, number of shoot/explant and number of leaves/ shoot compared with OM modified and MS modified media. Also, Zeatin at 4 mg L-1 recorded the highest shoot growth parameters during in vitro multiplication stage. During in vitro rooting stage, both the type and concentration of auxins have a significant influence on number of roots, root length and rooting % of Picual olive cultivar. In conclusion, it is recommended that very low concentrations of nano-silver particles be used in plants tissue culture on olive media (OM) supplemented with Zeatin at 4mgL-1.

Evaluation of different type containers used in micropropagation of Irish potato varieties

The Irish potato (Solanum tuberosum L.) is among the major crops grown in Rwanda especially in the Northern Province of Rwanda. In Rwanda, potato production and marketing chain still face serious problems including insufficient production and poor quantity and quality of seed material. The tissue culture was found to be a suitable method for the production of good quality and quantity of plantlets of Irish potato varieties. The experiment was performed in the tissue culture laboratory of RAB-Musanze station. The objective of the study was to evaluate the efficiency of the microbox in micropropagation of three varieties of Irish potato for safe seeds and rapid multiplication. The experimental design was laid on a complete randomized design with two factors; the containers with different filters and varieties (Kinigi, Kirundo, 393371-58). Parameters analyzed were the recovery rate, number of air roots, number of internodes and cuttings in containers and varieties. The results obtained showed a significant difference between containers on the recovery rate where the F-pr. < .001 at a significance level of 5 % and Kinigi variety (A) with Green filter has a high percentage. Considering the number of air roots, results showed that Kirundo variety (B) with Pink filter (3) had a high number of air roots. It was observed that 393971-58 variety (C) with Green filter (1) exhibited the highest number of internodes and a high number of cuttings. The studied parameters showed that the container with Green filter (1) performed well with 393371-58 variety (C) under aseptic conditions. The objectives of this research have been achieved and hypotheses verified and confirmed that “Under controlled conditions, the air rooting occurs more in the microbox than in the punnet and there is a difference of performance between containers and varieties while the recovery rate varies among varieties and type of containers. Moreover, it is necessary to continue the study on other varieties for a good evaluation of different type of containers and selection of the ones and variety that allow a good yield of quality plantlet of the Irish potato

The response of several combination of plant growth regulators to shoot induction of fig (Ficus carica L.) var. improved celeste

Improved Celeste is one of the common types of fig and potential to be cultivated in Indonesia. This research aimed to study the effect of several combinations of plant growth regulators on shoot induction of fig var. Improved Celeste. The research was conducted at Tissue Culture Laboratory, Department of Food Crops and Horticulture, Gedung Johor, North Sumatra, Medan. The research using a non-factorial Completely Randomized Design (CRD) with 4 treatments and 6 replications. The treatment of plant growth regulators (PGR) consisted of 4 levels, namely, MS media+ 0.5 BAP, MS media + 1 BAP, MS media + 0.5 BAP + 0.1 IBA, and MS media + 2 BAP + 0.2 IBA. The results showed that the best plant growth regulators in promoting shoot formation at the induction stage of fig var. Improved Celeste is MS media + 2 BAP + 0.2 IBA.

Effect of Paclobutrazol in micro tuberization of Potato (Solanum tuberosum L.) cultivar Granola Kembang and Repita

The seeds or seedlings are the main key to the success of potato cultivation. So one way to obtain high quality of potato seeds is to do with in vitro plant propagation or tissue culture. The research was conducted at Tissue Culture Laboratory, UPT Seed Horticulture Johor Building Medan, used the completely randomized design with two factors. The first factor was potato cultivar (K1: Granola Kembang, and K2: Repita), and the second factor was paclobutrazol M1 (MS + 0 ppm Paclobutrazol), and M2 (MS + 10 ppm Paclobutrazol). The results showed that the concentration of paclobutrazol significantly affected on the plant height, the number of shoots, the number of micro tuber, the micro tuber diameter, and the fresh weight of micro tuber.

Physiological character analysis of two shallot varieties at various watering frequencies

Analysis of the physiological characteristics of plants is important because the analysis includes various observations, calculations, as well as the varying relationships between plant growth and development processes and their results. The purpose of this study was to analyse the physiological characters of two shallot varieties at various watering frequencies. The research design used was a randomized block design with 2 factors, namely shallot varieties (Medan and Bima Brebes) and the frequency of watering (intervals of 1 once a day, once every 3 days, once every 5 days and every 7 days) located in the Greenhouse and Tissue Culture Laboratory of the Faculty of Agriculture, Universitas Sumatera Utara in May - September 2019. The results indicated that the Bima Brebes variety produced the highest total chlorophyll, which was significantly different from the Medan variety. The leaves relative water content was not significantly different in the two shallot varieties tested by watering frequency treatment. Watering once in 5 days produced the lowest hydrogen peroxide but produced the highest ascorbate peroxidase enzyme. The activity of the ascorbate peroxidase enzyme was significantly different and had the highest value in the Medan variety, watered with a frequency of once in 5 days.

The phenotypic appeal of lily (Lilium longiflorum Thunb.) prompted mutation by colchicine

One of the efforts to assemble high yielding varieties is to use a chemical mutation method, such as colchicine. The objective of this study was to examine the phenotypic appeal of lily convinced by colchicine to acquire a current application and interval of the period for soaked colchicine. The research was steered in the investigational research place of Institute for Vegetable Research (IIVR) at Tongkoh, Sub-District Dolat Rakyat, District Karo, North Sumatera, and Plant Tissue Culture Laboratory, Agriculture Faculty, Universitas Sumatera Utara, from July 2019 up to March 2020. The research castoff Randomized Block Design technique (Factorial-RBD) by two factorials, i.e.: application of colchicine (0, 100, 200, and 300 ppm) and the interval of time for soaked colchicine (3, 6, 9, and 12 h). The constraint experimental was the plant tallness, the span of bud after flourishing, and the width of bloom. The outcome of this study indicated the concentration of colchicine hip 300 ppm (K3) devours pointedly influenced on the plant tallness in the first week until sixth weeks when planting and the concentration of colchicine in 100 ppm (K1) has considerably influenced on the size of bud after flowering and the span of bloom.

Effect of Plant Growth Regulators on In Vitro Direct Organogenesis of Paulownia tomentosa plant

In vitro propagation of P. tomentosa (Thunb.) Steud. was performed in the Tissue Culture Laboratory of the Department of Agricultural Botany to find out the effect of plant growth regulators (PGRs) on the ability of stem internodes of Paulownia Plant to regenerate direct organs as well as on their morphological characteristics. Paulownia stem internodes obtained from seeds planted in vitro were cultured on Murashige and Skoog (MS) medium with supplements of different combinations of α-naphthalene acetic acid (NAA) with Benzyladenine (BA), and NAA with 2-isopentenyl adenine (2ip). The results showed that the combinations of 0.5 mg L-l NAA with 2 mg L-l 2ip, as well as 0.5 mg L-l NAA with 4 mg L-l BA gave the highest regeneration percentage, shoot length (cm), shoot fresh and dry weights (g). For root regeneration, the obtained shoots were excised and cultured on a rooting medium containing half-strength MS salts with different concentrations of indole-3-butyric acid (IBA) and NAA (each individually). It was observed that either the concentration of 1.0 mg L-l NAA or 2.0 mg L-l IBA resulted in the highest values of rooted shoots percentage, No. of roots per shoot, average root length (cm), and fresh and dry weights (g).

Fish Singgang Extracts as a Potential Anti-Proliferative against Colon Cancer Cell Lines (HT-29, HCT-116, CT-26)

Aim: To investigate anti-proliferative effect of three types of Terengganu singgang extracts on colon cancer cell lines (HT-29, HCT-116, CT-26).
 Study Design: Experimental study.
 Place and Duration of Study: Central Laboratory, Tissue Culture Laboratory, Universiti Sultan Zainal Abidin, Terengganu between April 2019 and July 2019.
 Methodology: Samples comprised three types of singgang dish, which were prepared, cooked, and then extracted with distilled water and ethanol (EtOH) in different strengths, 50%, 70%, and 100%.These singgang samples were chub mackerel (ST), Indian mackerel (SK),and a control sample with no fish (SC). Extracts were analyzed for their anti-proliferative effect by MTT-based assay. Then, the morphological of cell apoptotic changes was observed using light inverted microscope.
 Results: Experimental assays showed that the SC sample extracted in 100% EtOH produced the highest yield (3.7%).The extract of ST in aqueous (0.27 (0.11)) yielded the most cytotoxic value, followed by extract SK in 100% EtOH (0.28 (0.10)) and extract SC in 50% EtOH (0.20 (0.08)). Then, the anti-proliferative effect was confirmed with morphological changes of cell which were characterized by cell shrinkage, membrane blebbing, and fragmentation of apoptotic bodies after 24, 48 and 72 hours of treatment.
 Conclusion: In conclusion, the ST extract showed the best anti-proliferative effect.

In vitro The Role of Paclobutrazol in Potato Seed Production (Solanum tuberosum L) cultivar Granola Kembang Early Generation (G0) In Vitro

The aim of the research was to determine the best paclobutrazol treatment to obtain superior quality seed potato cultivars. The research was conducted at the Tissue Culture Laboratory of Horticulture Technical Centre Service Unit, Gedung Johor, Medan, North Sumatra, Indonesia from January 2020 to March 2020. The complete randomized design with a single factor, 4 treatments, and 6 replications was used in this research. The results showed that the concentration of paclobutrazol affected the number of leaves, the shoots length, the number of shots, the number of nodes, the number of internodes, the stem diameter, and the percentage of roots formed but not significantly affected the number of roots. The M2 treatment, MS + 9 ppm paclobutrazol, showed the best treatment with an average plantlet performance value of 7.33 and the plantlets showed the compact morphology with larger stem diameter and smaller thick dark green leaves.

Optimasi Konsentrasi Kinetin dan Benzyl Amino Purine Pada Kultur Tunas Vanili (Vanilla planifolia)

Vanilla has a potential to be developed through tissue culture techniques to anticipate the limitations of the parent plant as a source of planting material. The in vitro propagation ability of vanilla shoots needs to be controlled with the regulation of Kinetin and Benzyl Amino Purines. The interests of this study are 1) analysis of the response of vanilla explants at several Kinetin concentrations; 2) analysis of the response of vanilla explants at several concentrations of BAP and 3) analysis of the interaction of Kinetin and BAP on the response of vanilla explants to form shoot multiplication. The research was conducted at the Tissue Culture Laboratory Politeknik Negeri Jember from June to December 2020 using a factorial Completely Randomized Design (CRD). Factor 1 was the Kinetin concentration of 0.0, 1.0, 2.0 mg.L-1 and the second factor was the concentration of BAP 0.5, 1.5, 2.5 mg.L-1. The results proved that the fastest shoot multiplication occurred on MS medium + Kinetin 2 mg.L-1 with a mean of 8.7 days after inoculation. The mean number of shoots was 7.6 shoots/explant with the highest average wet weight of 0.9 grams/explant at the addition of BAP 1.5 mg. L-1 at measurement 70 days after inoculation.

In Vitro Conservation of Medicinal Plant St. John’s Wort (Hypericum perforatum L.) Through Dilution of Basic Medium

In vitro techniques could be applied for plant conservation through minimal growth. Growth reduction is generally attained by modifying the culture medium and/or the environmental condition. Conservation of St. John’s wort (Hypericum perforatum L.) in vitro through dilution of basic medium was conducted at the Tissue Culture Laboratory of Indonesian Spice and Medicinal Crops Research Institute (ISMCRI) in 2018. Sterile shoots were cultured into Murashige and Skoog (MS) medium by reducing macronutrients. The treatments used were full-MS + 0.1 mg L-1 N6 - benzyladenine (BA) as control; ¾ MS + 0.1 mg L-1 BA; ½ MS + 0.1 mgL-1 BA and ¼ MS + 0.1 mg L-1 BA. Observation of the culture growth was conducted three months after the treatments. The experiment was arranged in Completely Randomized Design with ten replications. The result showed that the use of a dilution of basic medium affected the growth of St. John’s wort. The use of ¼ MS + 0.1 mg L-1 BA composition could suppress culture growth (number of shoots, shoots length and number of leaves), without showing necrotic symptom until three months of conservation. This treatment could be used as an alternative to minimize the culture of St. John’s wort for in vitro conservation.

Effect of Added PPM on Murashige and Skoog Media for Shallot Meristematic Proliferation

Shallot (Allium ascolonicum L) are plants that belong to genus Allium sp that were propagated vegetatively through bulbs. In the developed countries, shallot seed production can be produced through in vitro/micropropagation. The activity observed the effect of explant source and added of PPM ( Plant Preservative Mixture) on Murashige and Skoog media in the growth and development of explants (Shoot tip, meristematic). The activity has been conducted in the tissue culture laboratory of IVEGRI from January to April 2019. As an explant bulb of shallot cv. Bima Brebes were infected OYDV or SYSV that has been tested by serology DAS ELISA. Media composition was MS (1962) with supplement sucrose 30 g L-1+ IAA 2 mg L−1 + Kinetin 2 mg L−1 + GA3 0.01 mg L−1 + Myo inositol 100 mg L−1 + gel rite 2 g L−1, pH 5.7. The treatment were PPM concentration (0;0.25;0.50;0.75; 1 mL L−1) and the explant : shoot tip (A), meristematic (B). Results of the experiment on visual observation were carried out randomly on growth and development, serology test of OYDV, and SYSV of the plantlets. On visual observation obtained MS media composition with 1 ml L−1 PPM the percentage of proliferation, there were no different from other concentrations of PPM and proliferation ≥ 60% for all media composition. DAS ELISA test for plantlet infected OYDV and SYSV for both explants was 47.37% - 69.33%.