The potential for biotechnological applications in crop improvement programs requires identifying genotypes that allow cell/tissue culture with predictable plant regeneration. In the past, many genotypes of wheat ( Triticum aestivum L.) have been examined for potential use in tissue culture studies. The present research work has also been designed to study in vitro callogenesis expression and regeneration potential of wheat cultivars under controlled laboratory conditions. Seeds of four elite commercial high yielding cultivars of wheat namely: NARC-2011, AAS-2011, PAK-2013 and GAL-2013, were collected from the Crop Science Institute National Agricultural Research Center (CSI-NARC) Islamabad, as the source of plant material for in vitro studies. The seeds were surface sterilized in 10% sodium hypochlorite solutions for 10 minutes with continuous shaking under laminar air flow hood. After that seeds were placed on MS (Murashige & Skoog, 1962) based callus induction and regeneration medium with various concentrations of 2, 4-D and BAP in separate test tubes. Maximum callus induction frequency of 90% for Pak-13 and AAS-11, followed by 87% and 83% for Gla-13 and NARC-11, respectively, was recorded at 4 mg/l and 6 mg/l of 2, 4-D. Similarly, maximum regeneration of 90% for AAS-11 and Pak-13, followed by 80% and 87% for NARC-11 and Gla-13 respectively, was recorded on MS basal medium containing 1.5 mg/l of BAP. An increasing trend in regeneration from 0.5 to 1.5 mg/l of BAP was observed but it gradually decreased with increasing concentration of BAP from 1.5 mg/l for all wheat cultivars. The callus formed under light was golden brown, dry nodule and smooth compact and less embryogenic while under dark conditions, it was white to yellowish white, dry nodule and compact and more embryogenic. Best results for callus induction and regeneration were obtained at temperature (24°C ± 1°C) for all wheat cultivars.
Wheat (Triticum aestivum L.) belonging to family Poaceae is the second most prominent grain crop in temperate countries and major staple food of the world [
Pakistan is an agricultural country and wheat is its most important cereal crop grown on 36% of total crop area. Conventional breeding methods in Pakistan are being used for enhancing the production and quality improvement of wheat crop. However, its average yield is very low and uncertain due to limiting constraints like wide range of biotic and abiotic stresses (drought stress, salt affected areas, aluminum toxicity, pests, weeds, and diseases); conventional plant breeding strategies are slow and less selective; restricted gene pool availability and species barrier in addition to other biological limitations are involved in decreasing the per hectare yield of wheat [
In recent years, biotechnology is playing important role in improving yield of many crops including wheat [
In vitro study pertaining to evaluate the efficient callusing and regeneration potentials of selected wheat cultivars was carried out in Plant Tissue Culture Laboratory, Department of Botany, Pir Mehir Ali Shah, Arid Agriculture University, Rawalpindi, Pakistan.
Seeds of four elite commercial high yielding cultivars of wheat namely, NARC-2011, AAS-2011, Pak-2013 and Gal-2013, were collected from Crop Science Institute National Agricultural Research Center (CSI-NARC) Islamabad as the source of plant material for in vitro studies.
The seeds of wheat cultivars were brought into the lab and were washed with running tap water. Then seeds were surface sterilized with 10% sodium hypochlorite (NaOCl) solution for 10 minutes with continuous shaking under laminar air flow hood. After this seeds were washed six times with autoclaved distilled water to remove the traces of sodium hypochlorite solution and then transferred on autoclaved filter papers in sterilized Petri plates for drying [
Ten seeds of uniform size of each cultivar were picked aseptically using forceps and placed on MS [
The frequency of callus induction and regeneration were recorded according to following formulas:
All in vitro culture were grown under ideal conditions of temperature, light and moisture.
Explant* = Seeds | Age of Cultures = 8 weeks | ||||||
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Tr. No | Media | Conc. (mg/l) | No of explants* cultured | No. of explants showing callus induction | |||
NARC-11 | AAS-11 | PAK-13 | GLA-13 | ||||
C1 | 1 | 10 | 1.3 ± 0.6 | 2.0 ± 1.0 | 3.0 ± 1.0 | 3.3 ± 1.5 | |
C2 | 2 | 10 | 4.7 ± 1.5 | 4.0 ± 1.0 | 4.7 ± 1.2 | 5.0 ± 1.0 | |
C3 | MS + 2, 4-D | 3 | 10 | 5.7 ± 1.5 | 6.7 ± 1.5 | 7.0 ± 1.0 | 6.7 ± 1.5 |
C4 | 4 | 10 | 7.0 ± 1.0 | 6.7 ± 0.6 | 9.0 ± 1.0 | 8.7 ± 0.6 | |
C4 | 5 | 10 | 6.3 ± 1.5 | 8.0 ± 1.0 | 7.7 ± 1.5 | 7.0 ± 1.0 | |
C4 | 6 | 10 | 8.3 ± 0.6 | 9.0 ± 1.0 | 8.7 ± 0.6 | 8.0 ± 1.0 |
Explant* = Seeds | Age of Cultures = 8 weeks | ||||||
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Tr. No | Media | Conc. (mg/l) | No of explants* cultured | No. of explants showing callus induction | |||
NARC-11 | AAS-11 | PAK-13 | GLA-13 | ||||
C1 | 0 | 0.5 | 10 | 2.7 ± 0.6 | 2.0 ± 1.0 | 5.0 ± 1.0 | 4.3 ± 1.1 |
C2 | 0 | 1 | 10 | 4.7 ± 1.5 | 4.0 ± 1.0 | 6.0 ± 1.0 | 5.7 ± 0.6 |
C3 | MS + BAP | 1.5 | 10 | 8.0 ± 1.0 | 9.0 ± 1.0 | 9.0 ± 1.0 | 8.7 ± 0.6 |
C4 | 0 | 2 | 10 | 7.0 ± 1.0 | 6.7 ± 0.6 | 7.0 ± 1.0 | 7.0 ± 1.0 |
C4 | 0 | 2.5 | 10 | 5.7 ± 0.6 | 6.7 ± 1.5 | 6.7 ± 1.1 | 6.3 ± 1.5 |
C4 | 0 | 3 | 10 | 5.0 ± 1.0 | 6.3 ± 1.5 | 6.0 ± 1.0 | 6.0 ± 1.0 |
The inoculated cultures were grown at different light and dark conditions. The light intensity at photoperiod of 16 hours light and 8 hours dark varied from 2000 - 3000 lux were tested.
The cultures were initially grown separately at 20˚C, 21˚C, 22˚C, 23˚C, 24˚C, 25˚C, 26˚C, and 27˚C in a growth cabinet (Hot pack) (
Each treatment was conducted with three replicates, and the results were presented as mean standard deviation. Data collected were analyzed statistically by using Student’s t- test to find out the variation between and within the treatments, with respect to percentage of callus induction and regeneration.
Various concentrations of 2, 4-D was used to check the callus induction in wheat cultivars. It was evident that the callus induction was triggered for all concentrations and among all cultivars, but the best results were observed on different concentrations in each cultivar (
Cultivars responded differently to concentrations of 2, 4-D in the media. All cultivars showed best callogenesis response at 4mg/l and 6 mg/l of 2, 4-D as compared to other concentrations (
Explant | Temperature (˚C) | |||||||
---|---|---|---|---|---|---|---|---|
20 ± 1 | 21 ± 1 | 22 ± 1 | 23 ± 1 | 24 ± 1 | 25 ± 1 | 26 ± 1 | 27 ± 1 | |
Seed | + | ++ | + + + | + + + | + + + + | + + + | + + + | ++ |
+ = Poor; + + = Fair; + + + = Good; + + + + = Excellent.
to different cultivars and differences in expression rate of callusing formation controlling genes. Yasmin et al. [
MS medium with varying concentrations of BAP were tested to check their effect on in vitro regeneration of wheat cultivars (
Highest regeneration 90% was recorded on MS basal medium containing 1.5 mg/l of BAP in wheat cultivars AAS-11 and Pak-13, followed by 80% and 87% in NARC-11 and Gla-13, respectively (
An increasing trend in regeneration from 0.5 - 1.5 mg/l of BAP was observed but it gradually decreased with increasing concentration of BAP from 1.5 mg/l in all wheat cultivars (
maximum regeneration was observed with MS medium supplemented with 0.5 mg/l BAP and 0.5 mg/l Kin [
Study of callus type, colour and texture is also important for identification of various responses of callus. The callus formed in light was golden brown in colour (
On the other hand callus induced in dark was white to yellowish white in colour (
Condition | Explant | Colour of callus | Type of callus |
---|---|---|---|
Light | Seed | Golden brown | Nodular/Smooth compact |
Dark | Seed | White to yellowish white | Nodular compact |
Effect of different temperature levels ranging from 20˚C ± 1˚C to 27˚C ± 1˚C was studied for callus induction and regeneration (
The present research work showed that high frequency callus induction and plantlet regeneration can be accomplished for selected wheat cultivars by using seeds as source of explants. The potential of callogenesis and regeneration in wheat cultivars is genotype and media dependent. Callusing and regeneration frequency of wheat cultivars can be enhanced to a considerable extent by using a combination of auxins and cytokinins in MS based medium. Optimization of these growth regulators, light and temperature conditions, is essential for maximum callus induction and plantlets regeneration in wheat cultivars. The results of present findings will be helpful for selecting the most tissue culture responsive wheat cultivars for further genetic transformation against different biotic and abiotic stresses as well as for improvement of important agronomic traits of wheat crop.
Iqbal, M., Raja, N.I., Asif, S., Ilyas, N., Hussain, M., Yasmeen, F., Ejaz, M., Sultan, M.A., Aslam, S. and Javed, H. (2016) In Vitro Study of Callogenesis and Regeneration Potential of Elite Wheat (Triticum aestivum L.) Cultivars. American Journal of Plant Sciences, 7, 2515-2526. http://dx.doi.org/10.4236/ajps.2016.717219