EFFECT OF PLANT GROWTH REGULATORS ON IN VITRO PLANT REGENERATION OF WHEAT

EFFECT OF PLANT GROWTH REGULATORS ON IN VITRO PLANT REGENERATION OF WHEAT (Triticum aestivum L.) FROM EMBRYO EXPLANTS
Doriana (Bode) XhulajInstitute of Plant Genetic Resources, Agricultural University of Tirana, Albania
[email protected]
EFFECT OF PLANT GROWTH REGULATORS ON IN VITRO PLANT REGENERATION OF WHEAT (Triticum aestivum L.) FROM EMBRYO EXPLANTS
Abstract
The experiment was carried in the laboratory of plant tissue cultures, of the Institute of Plant Genetic Resources, Agriculture University of Tirana. An efficient method was developed for multiple shoot regeneration of wheat from mature embryos without callus formation, for a short period of time. The effect of explants, isolated shoot embryonic meristem and shoot embryonic meristem with scutellum and various combinations of plant growth regulators in MS media on direct shoot regeneration of wheat was investigated. Among different combination of 2,4-D, IBA, NAA and BAP tested, embryo explants cultured in MS medium supplemented with 2 mg/L BAP, 0.6 mg/L 2,4-D resulted as the most efficient direct shoot regeneration and produces a maximum of 2.5 shoots per explants and 3.4 cm shoot length. Plantlets were successfully transferred to rooting medium. The greatest mean numbers of roots were obtained on MS media supplemented with 0.5 mg/L NAA, and the best mean value of root length (8.2cm) at 0.5 mg/L IBA. In this paper by simply manipulating the concentrations of BAP, 2,4 D, IBA and NAA in the culture medium, is described a method for rapidly obtaining whole plants of wheat without the subculture, using embryo explants.

Keywords: embryo, direct shoot regeneration, growth regulators, rooting
Introduction
Wheat (Triticum aestivum L.) is one of the leading cereals in the world. In Albania, it is the most important cereal crop and major staple food grown all over the country. Conventional breeding programs in Albania are used to enhance the production and quality improvement of the wheat crop. However, limited gene pool availability and long duration of these methods are the major limitations for improvement of the crop through conventional methods (Hamid et al. 2014).
Genetic engineering techniques are gaining popularity because the desired gene can be introduced from any source without the species barrier in the wheat genome in a short time to improve its characters (Malik et al. 2003). As suggested the transformation of the wheat crop entirely depends on the regeneration of transformed explants through tissue culture (Yu et al. 2008). Consequently, the establishment of reliable tissue culture protocols for plant regeneration is desired in order to improve wheat yield (Noor et al. 2009) through genetic transformation. Development of efficient plant regeneration protocol from either single cell or organized tissue is important for many commercially important crops like wheat the common source of energy and proteins for the world population (Hamid et al. 2014). Different authors suggest that tissue culture of wheat depends from their genotype (Mahmood et al. 2012), type of culture medium used (Mathias and Simpson 1986; Mahmood et al. 2012) and plant growth regulators (Saad et al. 2004). In vitro regeneration of wheat plants is achieved from different explants such as mature and immature embryos, seeds, endosperm, leaves etc. (Sarker and Biswas 2002). Among them, the immature embryo was reported as the best for callus induction and shoot regeneration (Sarker and Biswas 2002). But the availability of immature embryo is limited by wheat growing season or requires expensive and sophisticated growth chambers. On the other hand, mature seeds of wheat are readily available throughout the year, hence can be used for plant regeneration in any convenient time.
The present study was conducted to develop efficiently in vitro direct shoot regeneration by using mature embryos as explants sources. The objective of this research was tissue culture response of wheat explants grown on culture medium supplemented with different concentration of plant growth hormones. The results of the present study will be helpful for wheat breeding program through tissue culture technique and maintenance of desirable traits in wheat, also to avoid the risk of soma-clonal variation often observed in genetic manipulation process.

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Material and methods
Experimental site and source of explants: This study was carried in the Laboratory of Plant tissue culture of the Institute of Plant Genetic Resources, the Agriculture University of Tirana during 2016. Seeds of Albanian wheat (Triticum aestivum L.) cultivar Dajti used for this research are part of the seed collection of this Institute.

Seeds sterilization: mature seeds of selected wheat cultivar were washed under running tap water with detergent. Then they were disinfected with 75% ethanol for 30 seconds followed by 30% Clorox for 10 minutes with continuous shaking under laminar flow. Seeds were washed several times with autoclaved water to remove the sterilant.
Plant media: The mature embryos were aseptically excised from the caryopses, and then dissected to two types of explants including cut shoot embryonic meristem and shoot embryonic meristem with scutellum. Every explant was gently removed from the embryos and inoculated on Petri dishes with MS medium supplemented with different concentrations of plant hormones. The pH media was adjusted to 5.8±0.1 before autoclaving at 121 0C for 15 minutes. Cultures were kept in a growth room at the temperature of 25±10C for 2 weeks at photoperiod 8/16 h. After this period wheat explants were transferred at fresh medium for further proliferation and growth up to one week. After proliferation explants were transferred at regeneration media MS with different levels of BAP (1.0, 1.5, 2.0, 2.5, 3.0 and 3.5mg/L) in combination with IBA (0.5 and 1.5 mg/L), NAA (1.5 and 2.5 mg/L) and 2.4-D (0.4, 0.6, 0.8 mg/L) to get maximum regeneration and shoot induction. Each medium was supplemented with 3% sucrose, 1.5mg/L myo-inositol, and vitamins. Media were solidified with 6g/L agar. Plantlets were cultured at MS strong media with different concentration of auxin IBA (0.5, 1.0, 1.5, 2.0, and 2.5 mg/L) and NAA (0.5, 1.0, 1.5, 2.0, and 2.5 mg/L).When roots were developed plants were transferred in soil-filled pots. Data were collected for shot sprout frequency, shoot number, shoot length, leaves number, root number and root length.

Table 1. Plant growth regulators used for shoot induction and wheat plant regeneration
MediaPlant Hormones mg/L
BAP2,4-DNAAIBA
M1MS1.00.20.00.0
M2MS1.50.40.00.0
M3MS2.00.60.00.0
M4MS2.50.80.50.5
M5MS3.00.01.51.5
M6MS3.50.02.51.5
Effect of plant growth regulators on regeneration: Auxin and cytokinin are the most common plant growth regulators used in vitro culture of plant tissues (George et al. 2008). They turn the explants to produce a direct multiple shooting response (Malik et al. 2007), are important in cell division in differentiation stages and promoting shoot induction (Ganeshan et al. 2006). Reference (Ahmad et al. 2002) used a culture medium containing BAP and 2,4-D in meristem multiplication system for spring wheat genotype.
In this study the combination of BAP (2 mg/L) and 2, 4-D (0.6mg/L) was the best auxin/cytokinin ratio to conduct direct shoot regeneration. After two weak of maintenance in proliferation media, two types of embryonic explants used were transferred to regeneration medium with different levels of hormones. The survival rate was high (80%). According to the results of this study mature explants, cut shoot embryonic mersitem and shoot embryonic meristem with scutellum derived from Triticum aestivum L. embryos produced multiple shoots directly without intermediate callus phase on MS medium. Yasmin et al. (2001) observed that the mature embryos failed to initiate any type of calli at low concentrations of 2,4-D resulting only in initial swelling. Elimination of intermediate callus phase is an exclusive concession that used in rice (Nhut et al. 2007), cereal (Eudes et al. 2003) to decrease the time, frequency of somaclonal variation and especially genotype dependency (Sharma et al. 2004).
There was no significant effect of explants type on shoot regeneration in this study. Meristem section is one of the most important prerequisites for direct shoot regeneration. It was observed direct shoot regeneration without intermediate callus phase in all the explants after a short period (10 days). Isolated shoot embryonic meristem and shoot embryonic meristem with scutellum explants gave no significant differences in shoot regeneration, emphasizing the idea that the meristematic tissues have rapid cell division rate and represent a high potential for multiplication and regeneration, showing less variability in genotypic and phenotypic response, respectively (Eudes et al. 2003).

Figure 1. Embryo explants aseptically excised from the caryopses at wheat (Triticum aestivum L.) Dajti cultivar.Data given in Table 2, showed that the explants exhibited good response for regeneration in MS strong medium. Same results are reported by other authors (Raziuddin et al. 2010; Mahmood et al. 2012). They concluded that wheat cultivars have best response and perform better on MS while in another type of media used. Media was solidified with 6 g/L agar. The role of agar and its concentration on shoot regeneration has been object of many studies (Ali et al. 2004; Mahmood et al. 2012). It is thought that agar has agro pectin with its sulphate side groups and with some other organic impurities due to which it might have inhibitory effects on callus proliferation (Bhojani & Razdan 1996).
Table 2. Effect of plant growth hormones on multiple shoot induction of Triticum aestivum L. cultivar after 6 weeks of culture
MediaShoot sprout
frequency %Shoot number
(mean ±SE)Shoot length cm
(mean ±SE)Leaves number
(mean ±SE)
M1651.0±0.3c2.8±0.6bc2.4±0.3a
M2831.9±0.2b3.2±0.6ab2.1±0.4b
M3902.5±0.2a3.4±0.5a2.6±0.3a
M4852.1±0.2b1.9±0.6e1.7±0.4bc
M5701.4±0.5c2.0±0.5dc1.4±0.3d
M6621.7±0.3bc2.5±0.3cd1.6±0.4cd
The utility of BAP to induce multiple shoot formation was analyzed in a variety of experimental approaches to establish in vitro regeneration protocols using embryonic explants (Ahmad et al. 2002, Schulze et al. 2007). The results obtained in this study are consistent with those reported by Ahmad et al. 2002, where it is indicated that the higher values of BAP hormone in the media is capable of stimulating the differentiation of adventitious buds from shoot apices. In this study, the maximum values of parameters measured were observed in MS medium supplemented with 2 mg/L BAP (Figure 2; 2.5±0.2 shoot number; 3.4±0.5 cm shoot length and 2.6±0.3 leaves number).

Figure 2. Wheat multiple shoot formation
Rooting results: The micro propagated shoots were successfully rooted in full MS medium supplemented with either IBA or NAA concentrations. The greatest mean numbers of roots (3.1±0.2) were obtained on MS supplemented with 0.5 mg/L NAA. The highest value of root length (8.2±0.5) however was produced in IBA concentration (0.5 mg/L).
Table 3. Effect of auxin type on rooting of wheat regenerated plantlets after 6 weeks of culture
Type of auxinAuxin concentration mg/LRoot number (mean ± SE)Root length cm
(mean ± SE)
IBA0.51.3±0.2c8.2±0.5a
IBA1.01.4±0.2c7.7±0.6b
IBA1.51.4±0.3c6.9±0.6c
IBA2.01.2±0.2c5.6±0.8d
NAA0.53.1±0.2b4.7±0.4e
NAA1.03.7±0.2a2.1±0.4f
NAA1.53.5±0.3a1.9±0.2f
NAA2.03.3±0.3ab1.8±0.4f
The regenerated shoots were established well in the soil (Figure 3). The results obtained for rooting of the Dajti plantlets cultivar in MS media supplemented either with IBA or NAA goes in agreement with many studies.

Figure 3. Wheat (Triticum aestivum L.) of Dajti cultivar plants after acclimatization
Two types of auxin used in this study have been previously, successfully reported for inducing in vitro rooting in a variety of plants (Ramanayake et al. 2006; Mishra et al. 2008; Yasmin et al. 2009). In conclusion, the results of the present study for direct shoot regeneration and multiplication will be helpful for the wheat breeding program through tissue culture technique and maintenance of desirable traits in wheat, also to avoid the risk of soma-clonal variation often observed in genetic manipulation process.

CONCLUSIONS
The present study was conducted to develop efficiently in vitro direct shoot regeneration by using mature embryos as explants sources. The objective of this research was tissue culture response of wheat embryo explants grown on culture medium supplemented with different concentration of plant growth hormones.
Direct shoot regeneration is important since fewer soma clonal variations are likely to arise in the indirect regeneration method. The meristematic section is one of the most important prerequisites for direct shoot regeneration. In this study, it was observed direct shoot regeneration without intermediate callus phase, from isolated shoot embryonic meristem and shoot embryonic meristem with scutellum with various combinations of plant growth regulators in MS media, after a short period (10 days). Among different combination of 2,4 D, IBA, NAA and BAP tested, embryo explants cultured in MS medium supplemented with 2 mg/L BAP, 0.6 mg/L 2,4 D resulted in the most efficient direct shoot regeneration and produces a max of 2.5 shoots per explants and 3.4 cm shoot length. Plantlets were successfully transferred to rooting medium. The greatest mean numbers of roots were obtained on MS media supplemented with 0.5 mg/L NAA, and the best mean value of root length (8.2cm) at 0.5 mg/L IBA. In this paper by simply manipulating the concentrations of BAP, 2,4 D, IBA and NAA in the culture medium, is described as a method rapidly obtaining whole plants without the subculture of wheat using embryo explants.

The results of the present study will be helpful for the wheat breeding program through tissue culture technique and maintenance of desirable traits in wheat, also to avoid the risk of somaclonal variation often observed in genetic manipulation process.