Aseptic Culture Establishment Research Articles

Comparative study of different surface sterilization treatments and optimal month for establishment of aseptic cultures of raspberry cultivars

Comparative study of different surface sterilization treatments and optimal month for establishment of aseptic cultures of raspberry cultivars

Synergistic Effect of Plant Growth Regulators on Micropropagation of Eclipta alba: A Plant with Diverse Medicinal Properties

Synergism between various plant growth regulators is reported to be a key factor for the development of efficient in vitro propagation for any plant. Therefore, the current study examines the synergistic impact of plant growth regulators on in vitro propagation of Eclipta alba, an important medicinal plant possessing diverse medicinal properties. For the establishment of aseptic cultures, nodal segments were employed as explants on MS medium supplemented with 2.5 μM of 6-benzyle adenine (BA). Varying concentrations of BA and Kinetin (KIN)(0.0-5.0 μM), either alone or in combination with α- naphthalene acetic acid (NAA @ 0.0-5.0 μM) and indole 3-acetic acid (IAA@ 0.0-5.0 μM), were found to be effective for promoting shoot proliferation. Compared to KIN, BA was found to promote shoot proliferation and elongation more effectively. Further, the addition of 0.5μM NAA in the MS medium supplemented with 2.5 μM of BA increased shoot multiplication and elongation frequency from 58 and 17 percent to 65 and 21 percent respectively. The rooting frequency was found to be maximum on 1/2 strength MS medium supplemented with 5.0 μM of indole 3-butyric acid (IBA), which was found to be a superior auxin for inducing roots as compared to the NAA and IAA. With a 75% survival rate, in vitro raised plantlets were effectively acclimatized first in a poly house and later under greenhouse conditions. Molecular analysis was carried out using RAPD markers, with results indicating that the micropropagated plants were genetically identical to the mother plant. The developed micropropagation protocol for E. alba can be used at the commercial level for the mass multiplication of plants.

Optimization of micropropagation protocol and elicitation of luteolin and rutin biomolecules using phenylalanine and chitosan in root callus of Rumex hastatus D. Don

Rumex hastatus D. Don, a traditional medicinal plant is known for its antitumorous, antiangiogenic and antibacterial potential. HPLC analysis of different plant parts showed that root had the maximum quantity of luteolin and rutin and thus was selected for the establishment of aseptic cultures. Murashige and Skoog’s medium supplemented with 5 µM TDZ proved best for developing greenish-brown and compact calli in 98% root cultures within 30 days. Upon subculturing such callus pieces differentiated an average of 10.50 ± 1.29 shoots per culture. The excised shoots were transferred on MS + 0.1 µM IBA medium where they developed an average of 12.03 ± 2.49 roots per shoot. These plantlets were successfully acclimatized under greenhouse conditions and their genetic fidelity was evaluated employing SRAP and SCoT markers which revealed their high similarity with mother plant proving their true-to-type nature. Interestingly, the in vitro regenerants showed elevated levels of phenols, flavonoids, luteolin and rutin contents and higher antioxidant activities over the mother plant as revealed by phosphomolybdenum and DPPH assays. For elicitation, 100 mg/L phenylalanine was best for enhancing luteolin content (407.18 ± 10.58 μg/g d.w.) up to 17.42-fold over control (22.10 ± 0.57 μg/g d.w.) whereas, 25 mg/L chitosan proved most effective for accumulation of rutin content (1169.07 ± 46.49 μg/g d.w.) up to 2.74-fold over control (312.41 ± 12.42 μg/g d.w.). To the best of our knowledge, this is the first report on micropropagation and elicitation of luteolin and rutin in R. hastatus through root culture. The present investigation focused on selection of an elite plant part, regeneration through in vitro root callus, genetic fidelity analysis, evaluation of secondary metabolites, antioxidant activity and enhancement of luteolin and rutin using elicitors in Rumex hastatus.

Biotechnological approach for improvement of Crambe species as valuable oilseed plants for industrial purposes.

Boosting technological innovation for a sustainable and circular bioeconomy encompasses the use of renewable materials and development of highly effective biotechnological approaches to improve the quality of oilseed crops and facilitate their industrial deployment. The interest in cultivating Crambe as a potential crop is steadily growing due to its low propensity to crossbreeding with other oilseed crops, valuable seed oil composition and a high yield capacity. The main focus is located on Crambe abyssinica as the most adapted into the agriculture and well-studied Crambe species. At the same time, the Crambe genus is one of the most numerous of the Brassicaceae family featuring several underestimated (orphaned) species with useful traits (abiotic stress tolerance, wide range of practical applications). This review features progress in the biotechnological improvement of well-adapted and wild Crambe species starting with aseptic culture establishment and plant propagation in vitro reinforced with the use of genetic engineering and breeding techniques. The aim of the paper is to highlight and review the existing biotechnological methods of both underestimated and well-adapted Crambe species improvment, including the establishment of aseptic culture, in vitro cultivation, plant regeneration and genetic transformation to modify seed oil content and morphological traits of valuable species.

Sequential Sterilization of Banana (Musa Spp.) Sucker Tip Reducing Microbial Contamination With Highest Establishment Percentage

Sterilization procedure was standardized for grand naine cultivar of banana using various sterilants in combinations and alone. The observations were recorded regularly with respect to presence of fungal, bacteria as well as percentages of culture establishment. Results indicated that a treatment combination No. 3 [Lactic acid (0.15 %) + Tween-20 (0.1 %) + 0.8 % Commercial bleach (30 Min.) followed by Sodium chlorite (0.3 %) (20 min)] gave the highest percentage of aseptic culture establishment in vitro condition. The present study also showed that, single step aseptic inoculation was unable to control endophytic contaminants while sequential treatments were good enough to reduce microbial load as well as increase culture survival. Bangladesh J. Bot. 50(4): 1151-1158, 2021 (December)

In vitro shoot multiplication using meta-Topolin and leaf-based regeneration of a withaferin A rich accession of Withania somnifera (L.) Dunal

Withania somnifera (L.) Dunal is a popular medicinal herb used in traditional Ayurvedic medicines. The pharmacological properties of this plant are largely due to the presence of withanolides such as withanone, withanolide A, withanolide D and withaferin A. Among these, withaferin A has gained a lot of attention because of its anti-cancer activity against a number of cancer cell lines. The current study deals with the selection of an elite accession of W. somnifera with high withaferin A content and the establishment of aseptic cultures under in vitro conditions for clonal mass propagation. Here, a novel cytokinin meta-Topolin (mT) was used for the first time in W. somnifera for shoot proliferation. mT (2.5 μM) was recorded as an efficient cytokinin for successful shoot multiplication and their growth. Further, an effective and reproducible regeneration protocol using leaf explant of W. somnifera has been developed for in vitro mass propagation and genetic transformation studies. The best shoot regeneration frequency and the maximum number of shoot buds were recorded in MS medium amended with BAP (10.0 μM). Among various auxins such as IAA, IBA, and NAA, IAA (10.0 μM) was proved to be optimal for both shoot and root growth. The regenerated plants were transferred to the greenhouse for their hardening and a high rate of survival (80 %) was recorded. The genetic uniformity of in vitro regenerated shoots was verified with RAPD, ISSR, and SCoT markers.

Efficiency of the temporary immersion system versus the conventional in vitro propagation method

The work was carried out with the aim of assessing the efficiency of the methods of mass propagation of vitroplants through results obtained in the Conventional Method and the Temporary Immersion System (SIT) and achieve increases in the multiplication coefficient, in addition to characterizing both methods of in vitro micropropagation by describing them. Considering that conventional propagation methods would be slow to produce and of high production costs. This technique allows for proper planning, from the stage of establishment of aseptic culture in the laboratory, to the establishment and production of the crop in the field. However, in temporary immersion crops, the constitution and physiological proceeding of outbreaks are very similar to those shown in ex vitro environments, due to the evolution of the system environment. Several authors, including Cruzat, (2009), states that the use of THE SIT reduces production costs per plant; this as a result of the mechanization of some stages of micropropagation and Winkelman, et al, (2006), commenting as in bioreactors, obtaining seedlings allows the reduction of production costs by between 50 and 60 %. Results that corroborate the efficiency of the temporary immersion system against the conventional in vitro propagation method.

In vitro culture and adventitious rhizogenesis of Bambusa tulda Roxb. in agro-climatic conditions of Bihar, India

Bambusa tulda is highly economical bamboo, playing an important role as non-timber raw material for large number of products. Forest resource of this bamboo is depleting at a high rate and hence to meet its growing demand in commercial sector, technique for industrial production is required. The present experimental studies were carried out for rapid and mass clonal propagation of B. tulda covering two major aspects of propagation i.e. estab-lishment of aseptic culture and adventitious rhizogenesis. Organogenetic mode of regeneration of plant tissue culture technique has been employed for massive production of this bamboo species. The treatment of explant with Dettol (0.4%) for 10 mins and Bavistin (0.2%) for 30 mins followed by Ethyl alcohol (70%) for 35 seconds and HgCl2 (0.1%) for 10 mins resulted with maximum number of survival of aseptic cultures. The effect of MS medium+ additives+ NAA (0.25mg/l) supplemented with TDZ (0.5mg/l) showed maxi-mum (96%) shoot initiation. Maximum root growth (8.8cm) and number (approx 7 roots/ clump) was observed in ½ strength of MS with IBA (4mg/l) supplemented with coumarin (4mg/l). Seventh sub-culture cycle/ passage was the most suitable and the earliest possible passage of cultures (sub-culturing) for generation of profused in vitro rooting. The establishment of aseptic in vitro cultures and profused adventitious root induction derived from above studies will further help in industrial production of quality planting material of B. tulda within short span of time.

Production of quality planting material of Chandler strawberry by in vitro regeneration

In order to explore the in vitro regeneration potential of strawberry, experiments were conducted on MS medium supplemented with different concentrations of cytokinins and auxins alone and in combination for establishment of aseptic cultures, shoot regeneration and multiplication, rooting and acclimatization of plantlets. Two type of explants viz., runner tip and nodal segment were used for the investigation. Establishment of aseptic cultures is a pre requisite for successful culture. Among several sterilizing agents ethanol (70%) for 30 sec followed by HgCl2 (0.1%) for 2 min was found to be the best treatment resulting in least contamination and maximum survival (53.3 ± 1.08 percent in runner tip and 53.1 ± 1.08 percent in nodal segments). Phenol exudation was the major problem during establishment which caused death of most of the explants. In our experiment, explants were inoculated in MS medium supplemented with ascorbic acid 200 mg per liter to control browning. Addition of BAP alone or in combination with IAA was found essential during shoot proliferation and multiplication. Among all the treatments, 3.0 mg/L BAP + 0.5 mg/L IAA was found most effective with regard to minimum number of days (16.1 ± 0.29) required for shoot induction and maximum number of shoots (12.8 ± 0.52). Half strength MS media with 1.5 mg/L IAA was found superior over other concentrations of IAA, IBA and NAA for various rooting parameters. During acclimatization, maximum percentage survival (45.2) was recorded when coco peat alone was used as primary hardening after 20 days and combination of coco peat with FYM (1: 1 v/v) recorded maximum percent survival (65.0) after 30 days in secondary hardening.

In vitro culture and diversity of endophytic fungi in Bambusa oldhamii

ABSTRACT Bambusa oldhamii Munro is a fast-growing species of woody bamboo with strong commercial appeal. In Brazil, the use of this species is limited, mainly due to the low availability of seedlings for commercial plantations. Micropropagation is a technique used for the large scale production of seedlings, but protocols for the establishment of aseptic cultures are hampered by the presence of endophytic contamination. This study aimed to develop an in vitro establishment protocol for B. oldhamii, as well as to make the molecular identification of fungi associated with the explants used. Nodal segments of adult plants grown in the field were used as explants. This material was submitted to two experiments carried out to evaluate the effect of 6-benzylaminopurine (BAP) on multiplication and of Plant Preservative Mixture (PPMTM) as a disinfectant. In the first one, 10 µM, 15 µM or 20 µM of BAP were combined with 1 mL L-1, 2 mL L-1 or 3 mL L-1 of PPMTM; while the second one used 0 µM, 2.5 µM, 5 µM or 7.5 µM of BAP with 4 mL L-1 of PPMTM, both added to MS culture medium. After 21 days of culture, the use of 4 mL L-1 of PPMTM inhibited the bacterial growth and reduced fungal contamination. The addition of BAP to the culture medium above 10 µM inhibited the formation and growth of new shoots, while additions of less than 7,5 µM had no effect. The molecular identification of the endophytic fungi isolated during the in vitro culture indicated the presence of numerous fungal species, increasing the current knowledge about the diversity of fungi associated with bamboo.

In Vitro Clonal Propagation of Musa Sp. Cultivar Gopi: A Palatable Banana of Tripura, India

The present investigation has been concentrated on the establishment of an efficacious method for <i>in vitro</i> regeneration of a <i>Musa</i> cultivar Gopi of Tripura. Focus has been greatly anchored to the sterilization procedure for assuring successful aseptic culture establishment of this cultivar. The culture protocol for shoot induction and proliferation comprised of four different treatments in basal MS medium fortified with 6-Benzylaminopurine of varying concentrations. The optimum response was encountered in the treatment of BAP in MS medium at concentration of 8mg l^-1 for shoot bud induction and multiplication. Subsequent shoot growth and multiplication was achieved through repeated subcultures in media containing lower concentration (4 mg l^-1). Root initiation was stimulated and brought about by treatments with two different concentrations of Indole butyric acid. The best rooting was manifested in treatment with IBA at concentration of 2mg l^-1. The present study is an approach towards successful establishment of a simple and rapid clonal propagation of a potent Musa cultivar Gopi having traditional and commercial value from this region of Tripura, North-east India.

Studies on Establishment of Aseptic Culture in Pomegranate cv. Bhagwa

Studies on Establishment of Aseptic Culture in Pomegranate cv. Bhagwa

An improvised in vitro vegetative propagation technique for Bambusa tulda: influence of season, sterilization and hormones

Season and concentration of sterilizing agents play a significant role for establishment of aseptic in vitro shoot cultures and sprouting of nodal explants from field growing culms of bamboo species. In the present investigation the nodal segment explants of Bambusa tulda Roxb collected in different seasons and treated with various concentrations of HgCl2 showed significant variation in aseptic culture establishment and bud break. The rainy season (July–August) recorded with highest of 78% aseptic culture establishment whereas autumn recorded with lowest 46%. Summer and winter seasons emerged to be the best period, registering > 60% in vitro bud break. On the other hand, the autumn season had the lowest value for bud break, i.e. 42%. Among different doses of sterilizing agent tried, HgCl2 0.1% found to be suitable for maximum aseptic culture establishment (66%) as well as bud break (59%). However, among the interactions, summer season and the dose of 0.1% HgCl2 exhibited maximum of 73% response for both aseptic culture establishment and bud break. MS medium (liquid) enriched with 5.0 µM BA + 5.0 µM Kn [Kinetin (N6-Furfuryladenine)] with additional supplementation of 100 µM glutamine + 0.1 µM IAA supported a maximum in vitro shoot multiplication of 4.75 fold. The proliferated shoots were successfully rooted on MS medium (liquid) supplemented 40 µM coumarin. The plantlets transferred to the polythene bags showed 98% survival.

English

In vitro propagation of banana preferably use sword sucker as explant source where microbial contamination poses a great problem in establishment of aseptic cultures. This study demonstrates up to 36% reduced microbial contamination in aseptic culture establishment and subsequent micropropagation due to osmotic stress induction in the banana suckers. Osmotic stress was induced by keeping the freshly collected suckers in shade and measuring fresh weight at 0, 7, 14, 21, and 28 days interval to ascertain loss of moisture. Stress induced for 21 days showed 58.85% moisture loss showing lowest contamination upto 40% against 76% for fresh suckers. Micropropagation of Musa sp. (Malbhog variety) through shoot tip culture of stressed suckers was carried out in Murashige and Skoog (MS) medium supplemented with 1 to 2 mg/l &nbsp;6-benzyl-aminopurine (BAP), 2 to 4 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l &nbsp;&alpha;-naphthalene acetic acid (NAA). Multiplication of plantlets was observed till 6th passage. From 19 aseptically established explants of 21 days stressed sucker sub cultured in MS medium fortified with 1.0 mg/l BAP and 0.5 mg/l NAA, for 6th passage produced 5122 plantlets. Regenerated micro shoots were rooted in MS medium fortified with 0.5 mg/l IAA. The plantlets were hardened in polybag containing soil and seasoned cow dung. &nbsp; Key words: Osmotic stress, shoot tip culture, in vitro, Musa, Malbhog.

An efficient micropropagation protocol forStevia rebaudiana

The present study was conducted to develop an efficient and reproducible mass multiplication protocol in stevia. The shoot-tip and nodal segments were used as explants. The survival percentage of nodal explants was 100%, when explants were treated with HgCl2 (0.1%) for 3 minutes along with bavistin (0.2%) and streptocyclin (0.2%) for 45 minutes. The nodal and shoot-tip explants were best responded to MS medium supplemented with 2.0 mg/l BAP. Among the different media used, MS medium supplemented with 2.0 mg/l BAP showed the maximum shoot induction (98.0%) in 4.7 days and (74.0%) in 7.1 days using nodal and shoot tip explants, respectively. BAP alone was found to be better for establishment of aseptic culture of stevia instead of combinations of different cytokinins and auxins. MS basal medium supplemented with 0.3 mg/l BAP + 0.3 mg/l Kin + 0.1 mg/l NAA was proved to be the best with 34.9 shoots/explant developed from nodal and shoot-tip explants after 30 days on culture medium. Maximum rooting (90±7.75%) was observed on the half MS medium supplemented with 0.5 mg/l NAA with 21.2 roots per shoot in 7.4 days. The roots formed in medium without hormone were not healthy. Rooted shoots were separated individually and transferred to plastic bag containing sand, soil and vermi compost in ratio of 1: 1: 1 with 100% survival. The hardened plants were transferred to field using recommended agronomic practices.

Micropropagation of grapevine (Vitis vinifera L.) seedless cultivar 'Parvana' through lateral bud development

Factors affecting successful establishment in vitro, rapid proliferation, rooting and acclimatization of grapevine seedless cultivation 'Parvana' were studied. A treatment combination of 2.0 % calcium hypochlorite for 7 minutes plus 70 % ethanol for 3 minutes was found to be effective with 75.0 % of aseptic culture establishment in vitro. The highest number of new microshoots (4.5) was obtained in a culture medium (Gambourg's B5 modification) supplemented with 0.6 mg∙L-1 BAP + 0.2 mg∙L-1 KIN + 0.5 mg∙L-1 GA3. Presence of GA3 in combination with BAP and KIN promoted elongation of shoots. A concentration of 0.4 mg/l IAA in half strength of MS (Murashige and Skoog) proved to be the best for rooting (84.4 % root induction, 4.66 roots with 7.9 cm length). In vitro rooted plantlets were successfully acclimatized, with 82.2 % survival rate in plastic pots containing garden soil, sand and peat moss(1:1:1). In this study an efficient micro propagation technique of grapevine seedless cultivar 'Parvana' was successfully achieved through lateral bud culture. Such a technique would be useful for large scale plant production and in vitro conservation.

Optimization of Factors Affecting In vitro Establishment, Ex vitro Rooting and Hardening for Commercial Scale Multiplication of Silk Banana (Musa AAB)

Factors concerning aseptic culture establishment and hardening were studied in detail in choicest Silk Banana. Effect of size of sucker (small, medium and large), carbon concentration (10, 20 and 30 g/l), season of initiation (wet and dry) and pre-treatments such as segmentation and incision to the explants were studied during initiation. Further, hardening related factors such as substrates used for primary and secondary hardening, nursery nutrition (source and frequency of application) and pre-treatments for ex vitro rooting were also studied. Results revealed that small suckers were most suited for initiations with the least contamination, maximum establishment and higher percentage of greening. Lower concentration of sucrose, though delayed greening, resulted in 100 % establishment of explants. Initiations performed during the drier period were completely free from the fungal contamination and showed less bacterial contamination than those performed during the rainy season. Segmentation of explant into four parts during first subculture supported maximum shoot proliferation by overcoming apical dominance. Coir pith was observed to be the most congenial substrate during primary hardening, whereas coir pith alone or sand: red earth: coir pith (1:1:1) supported superior performance of plantlets during secondary hardening. Single application of mono ammonium phosphate improved growth of plantlets during secondary hardening. Pre-treatment with mono ammonium phosphate gave the best response in terms of rooting and hardening of un-rooted micro-shoots. Thus, the discussed methodology could help the industries to take up commercial scale propagation of Silk Banana.

Rapid Propagation of Sweet and Sour Cherry Rootstocks

The paper presents a protocol for micropropagation of Prunus sp. rootstocks included in the sweet and sour cherry breeding program. Germplasm diversity for rootstock breeding derives from natural populations, where conditions and biological vectors for systematic infection with viral diseases are constantly present. The establishment of aseptic culture depends primarily on the explant type, as all selections were collected from natural habitat. For nearly all investigated selections, dormant buds were the favored source, due to enabling rosette initiation in more than 58% cases. In P. cerasus L. selections, 100% contamination was noted when shoot tips were used as an explant source. Significant influence of the double-phase medium on the number and height of multiplied shoots was observed in the standard cherry rootstock, ‘Gisela 6’. For P. fruticosa Pall., selection ‘SV1’ and ‘SV2’, and P. cerasus ‘D6’ selection, the double-phase medium also had a significant effect on the height of multiplied shoots, when compared to solid DKW (Driver and Kuniyuki Walnut) medium. Genetic variability of selections within the investigated species resulted in variable plant rooting success. Adding Fe-EDDHA (Ethylenediamine di-2-hydroxy-phenyl acetate ferric) in the 200 mg l-1 concentration to the rooting medium significantly enhanced the percentage of rooted plants. The highest rooting percentage was noted for ‘Gisela 6’ and ‘D6’ genotype at 1 mgl-1 IBA (indole-3-butyric acid), while 0.8 mgl-1 was the optimum concentration for P. mahaleb L. ‘M1’ selection. P. fruticosa genotypes required significantly higher IBA concentration for rooting (2.5 and 3.5 mg l-1).

Aseptic Culture Establishment Using Antibiotics with Reference to Their Efficiency and Phytotoxicity in Difficult-to-Establish Native Ney Poovan Banana (Musa, AB)

Native banana varieties possess a large number of agronomically superior traits and hence, have a great potential in the domestic as well as international market. Though, India is considered as home of bananas, not many varieties have been grown on commercial scale. Micropropagation, which offers a reliable tool for multiplication of plants, could be of great use in producing quality planting material in such underexploited varieties, provided an efficient protocol is available. Present study is aimed at standardizing antibiotic supplement for obtaining aseptic cultures in difficult-to-establish native banana variety, Elakki Bale. Of the different antibiotics and combinations tried, chloramphenicol was found to give 100 % aseptic cultures followed by rifampicin + chloramphenicol, rifampicin and chloramphenicol + streptocycline combination. Rifampicin was found to cause least phytotoxicity. The plantlets grew normally in subsequent cycles and 96.3 % plantlets could survive upon transfer to ex vitro conditions.

Regeneration of phytoplasma-free Artemisia roxburghiana Besser var. purpurascens (Jacq.) Hook. plants using apical meristem culture

We have used apical meristem culture to develop an efficient protocol for reducing phytoplasma infection of Artemisia roxburghiana Besser var. purpurascens (Jacq.) Hook. plants. Shoot tips of different sizes from phytoplasma-infected field-grown plants were treated with 0.1% mercuric chloride (HgCl2) for 1 min followed by a 30-s exposure to 70% ethanol. The size of the explants significantly influenced the survival frequency and the success of aseptic culture establishment. Sterile explants responded notably to 13.95 μM Kinetin (Kn) and 0.27 μM α-naphthalene acetic acid (NAA), and a maximum of 38 ± 0.87 shoots per explant could be obtained after 6 weeks of incubation. Sub-culturing of the shoot mass after 8 weeks of culture on the previously described medium to 8.88 μM 6-benzyladenine (BA)- and 0.27 μM NAA-containing medium stimulated further multiplication, elongation and growth of each individual regenerant. Efficient rooting was noted after 5 weeks of transfer on half-strength MS medium containing 4.93 μM IBA. Sequential hardening as hydroponic cultures under culture room and glasshouse conditions led to almost 85 and 98% survival of the regenerants upon transfer to pots and field, respectively. Unlike the original A. roxburghiana plants, the plants raised from tissue culture showed a total absence of inherent phytoplasma infection evaluated via inspection of morphological features, PCR and microscopic observations.