Surface Sterilization Of Explants Research Articles

Development of a Suitable in vitro Regeneration Protocol for Rahangala Pear (Pyrus pyrifolia L.) in Sri Lanka

The limitation of planting material is the major drawback to expanding pear cultivation in Sri Lanka. Hence, an in vitro protocol was developed to produce planting material for the pear variety of Rahangala. Pear shoots were used as explants and surface sterilization of explants was done using 0.5% and 1% (w/v) AgNO3 for 15 min, 5% and 10% (v/v) NaOCl for 5, 10 and 15 min. In vitro shoot multiplication and root induction were found to be suitable in full and half strengths of MS medium respectively supplemented with various concentrations of BAP (1, 1.5, 2, 2.5, 3 mg/l), IBA (0, 0.5, 1, 2, 3, 4 mg/l) and NAA (0.1 mg/l). Acclimatization was conducted in pots containing different proportions of topsoil, compost, coir dust and sand. Completely Randomized Design (CRD) was used as an experimental design for this study. During sterilization of explants, the survival rate of 86.6% was recorded after 3 weeks where the treatment was done with 1% AgNO3, and 10% NaOCl for 10 min. BAP (3.0 mg/l) produced the best shoot multiplication (1.46 ± 0.2). Significantly (p<0.05), the highest mean number of roots (1.93 ± 0.98), average length (60.6 mm), and rooting percentage (68%) were observed in ½ MS medium with 4.0 mg/l IBA. After five weeks of transplantation in pots, 100% of plantlets survived in topsoil, compost, coir dust, and sand in a ratio of 1:1:1:1 Plant Tissue Cult. & Biotech. 34(1): 25-33, 2024 (June)

Identification of Best Exposure Timings of Sodium Hypochlorite in Surface Sterilization of Different Explants in invitro Propagation of Annona muricata

The most crucial and delicate stage in plant tissue culture is the surface sterilization of the explant. An inappropriate concentration of sterilant may have deadly effects on explants by altering cell division and other metabolic processes that limit the growth and development of explants. Considering the explants observations and sterilization treatments of Sodium hypochlorite (4%), T6 (7 min), T7 (8 min) and T8 (9 min) are selected for green nodal explants as highest response, least days for initiation of response, good health with less contamination and moderate survival of explants. Whereas, T5 (6 min) T6 (7 min) and T7 (8 min) are selected for leaf with lower contamination with good health and survivability. Internode was not selected due to no response with higher contamination and less survivability. Brown nodal explants responded very less and less survivability with higher contamination as compared to other explants

Optimizing Surface Sterilization for Micropropagation of Gerbera jamesonii: A Study on Explant Survival and Contamination Control

In the commercial production of gerberas, micropropagation enables the large-scale production of plants in a limited space, as well as the obtaining of disease and pest-free uniform plants. This technique also ensures precision in production schedules and product quality, regarding plant homogeneity and vigour. Nevertheless, the occurrence of contamination in an in vitro culture is frequent, and might result in high damage. Plant materials have different kind of microbes i.e., exogenous and endogenous in nature which is the main reason for contamination in tissue culture. Pre-treatments to explants with fungicides and bactericides can be effectively used to eliminate the surface contamination. Surface sterilizing agents, their levels and duration of exposure are known to impact the in vitro culture establishment. In the present experiment, pre-treatment of explants was carried out with Teepol BleachTM solution (0.1%) and BavistinTM (0.1%) for 15 and 20 minutes respectively for effective control of fungal contaminations in all the cultures. For the control of bacterial contamination antibiotics were added in culture medium. Surface sterilization of explants with 0.1% mercuric chloride (HgCl2) for five minutes gave the maximum survival of the growing cultures.

A Fumigation-Based Surface Sterilization Approach for Plant Tissue Culture.

Plant tissue culture has led to breakthroughs in understanding and applying the fundamental knowledge towards harnessing more from plants. Microbial contamination is one of the serious problems limiting the successful extrapolation of plant tissue culture practices. Sources of in vitro contamination include culture containers, media, explants, equipment, the environment of the culture room and transfer area, and operating personnel. The successful initiation of in vitro culture mostly depends on surface sterilization of explants because this is the primary source. Usually, surface sterilization is done using chemicals, or toxic nanomaterials, this is the first time such an approach has been demonstrated. Numerous surface microflora attached to plant surfaces grow faster than the cultured explants and release phytotoxic substances into the culture media, hindering positive outcomes. In the current work, for the first time, the applicability of turmeric and benzoin resin-based fumigation of explants is demonstrated. The results showed that fumigation methods for surface sterilization were promising and could lead to fifty and even 100% contamination-free plant tissue culture. Nanoparticulate carbon was identified in the turmeric and benzoin smoke and coined the key player in the surface sterilization effect. These studies open a whole new avenue for the use of fumigation-based methods for riddance of microbial contamination.

Effect of explant type and surface sterilants on asepsis and survival culture for callus induction in peach

For the optimization of surface sterilization of explants and their in vitro establishment for callus, aseptic culture and survival of explants were measured. The present study was carried out to investigate the influence of explant type, various surface sterilants and their exposure time on the establishment of explants for callus induction in peach (Prunus persica L. Batsch). The leaf disc and intermodal segment explants taken from newly growing twigs of peach cultured on MS medium supplemented with BAP (0.5 mg/L) + 2, 4-D (2 mg/L) for callus induction after surface sterilization with various surface sterilents. The overall results suggested that the best sterilization treatment for culture establishment in peach explants was sequential exposure of explants to ethanol (70%) 30 second + HgCl2 (0.1%) 3 minute + NaOCl (1.0%) 3 minute resulting 63.34 per cent aseptic culture with 80.00 per cent survival of explants in both leaf disc and internodal segment comparatively suitable among all the five treatments. Further increase in exposure time to various sterilants resulted in significant reduction in survival of explants. Hence, ethanol (70%) 30 second + HgCl2 (0.1%) 3 minute + NaOCl (1.0%) 3 minute was used for surface sterilization of explants in peach for establishment of explants for callus induction.

In vitro propagation of almond (Prunus dulcis) cv. Merced

Forced and unforced shoot tips were surface sterilized with different sterilant regimes and incubated under normal culture room conditions. Surface sterilization of explants with mercuric chloride 0.1% (w/v) for 10 min. was found effective in improving culture asepsis (51.66%) and explant survival (55.00%). Higher values for both these parameters were recorded with forced explants in comparison to unforced ones. Main effect of growth regulators and media was significant on explant establishment which was maximum (66.66%) on ½ MS media containing BAP + IBA (0.50+0.01 mg/l). Callusing at the base of initiating cultures was minimum (24.58%) with BAP+IBA (0.25+0.01 mg/l). Microshoots from the established cultures were subcultured on the MS media supplemented with BAP and NAA alone or in combination for axillary shoot proliferation. Maximum proliferated cultures (86.66%) with maximum shoot number/explant (15.61) and proliferation grade (4.00) was obtained with BAP+NAA ( 0.40 + 0.01 mg/l). BAP was found superior to NAA during axillary shoot proliferation. Microshoots (10–15 mm) from proliferated cultures were subcultured in root induction medium (MS medium supplemented with IBA) and incubated under darkness for 10 days at 24±1 oC and then transferred to root development medium (hormone-free MS medium) and incubated under normal culture room conditions. Highest rooting of microshoots (93.33%) with maximum root number/shoot (5.90) and root length (43.00 mm) was obtained with IBA (1.0 mg/l).

In vitro callus induction protocol for Leptadenia pyrotechnica using various explants

An efficient protocol has been developed for a rapid callus induction in Leptadenia pyrotechnica. Nodal, internodal and pod explants from mature plant of L. pyrotechnica were cultured after surface sterilization on Murashige and Skoog medium supplemented with plant growth regulators (PGRs) cytokinins and auxins individually and with various combinations. Nodal segments proved the best explants (90% callus induction) compared with internodal and pod explants (5% and no callus induction respectively). Different treatments were employed for surface sterilization of explants revealing that combination of sodium hypochlorite (NaOCl2) and mercuric chloride (MC) were found significant. Minimum contamination (7%) occurred at 30% NaOCl2 + 2 g/L MC, while 80% occurred at 30% NaOCl2 + 1 g/L MC. The nodal segments cultured on MS medium supplemented with 0.5 mg/L NAA that produced maximum callus (90%) within four weeks. It was followed by 60% callus induction at 5 mg/L NAA + 2.5 mg/L benzyl-adenine (BA) and 30% at 10 mg/L 2,4-D + 3 mg/L 2ip + 3 g/L activated charcoal (AC), while only 20% appeared at control (without hormone application). This is the first successful attempt to establish consistent callus formation protocols from nodal stem segments of L. pyrotechnica. This study may contribute in conservation management of this native plant species found especially in the deserts of Pakistan. Key words: Callus induction, Leptadenia pyrotechnica, plant growth regulators, cytokinins, auxins.