Establishment of an efficient regeneration protocol of six different varieties of wheat ( Triticum aestivum L.)

An efficient callogenesis and regeneration protocol was optimized for six different wheat (Triticum aestivum L . ) varieties including Chakwal-50, Galaxy-01, NARC-09, Pakistan-13, Millat-11

and nutrients to more than two billion people and have essential role in food security as it facilitate with 20% of world calories (Bhatta et al., 2017).Conventional breeding faces two major limitations, that is, restricted gene pool and extended duration.Simultaneously, wheat genome size is ~17000 Mb, which is challenging to improve genetically.As compared to traditional breeding, robust techniques of biotechnology such as CRISPR/ Cas9 and other most advanced technology to modify DNA with addition or deletion of nucleotides are more precise and encompass all challenges (El-Sappah et al., 2021), additionally, inter-and intra-species transmission of stress associated traits is also possible with a shortest duration (Singh et al., 2021).To employ aforementioned techniques, a robust and highly efficient regeneration protocol is mandatory (Yu et al., 2008).
Dedifferentiation of cells into tissues and altering gene expression pattern within a cell are basic need for regeneration of plants in controlled environment (Bull and Michelmore, 2022).In-vitro regeneration of plants happen by three different pathways including repairing of tissues, clonal embryogenesis, and de novo organogenesis (Long et al., 2022).First pathway of tissue repairing involves the process of regeneration of tissues after roots, shoots and tips of leaf get wound or cut, and this mostly used in those techniques that utilized non-reproductive plant parts (Xu and Huang, 2014).In cell, tissue and organ cultivation techniques, plants are mostly regrown through non-reproductive cells as well as through de novo organogenesis (Hill and Schaller, 2013).De novo organogenesis can occur through two ways including direct and indirect de novo organogenesis.Direct de novo organogenesis takes place naturally while indirect de novo organogenesis takes place in-vitro.At the time of de novo indirect organogenesis, cells of explants pass through dedifferentiation and different plant growth regulators including auxin and cytokinin activate cell division (Sugimoto and Meyerowitz, 2013).Numerous explants of wheat namely seed, immature as well as mature embryo, endosperm, root tips and shoot apical meristem can be employed for in-vitro formation of callus and tissues regeneration (Kumar et al., 2017;Mahmood and Razzaq, 2017).Among all, young and immature embryos are best choice (Kumar et al., 2017).For example, immature zygotic embryos of barley (Hordeum vulgare L.) displayed significantly high callogenesis and regeneration (Abbas et al., 2023).In order to develop heat stress resistant maize verities (El-Sappah et al., 2022), immature embryos of maize (Zea mays) were employed to induce callogenesis and regeneration, which resulted in significant regeneration rate (Wei et al., 2022).Nonetheless, pre-mature embryos are not accessible consistently throughout the year.Contrastingly, mature seeds are available around the year, therefore, considered best choice (Rahman et al., 2008).In this study, we employed mature seeds in callogenesis and regeneration to investigate their potential.
Murashige and Skoog (MS) media containing specific concentration of hormones for accelerating growth such as 2,4-D, IAA, and Kinetin is promising media for plant callogenesis and regeneration.MS supplemented with 200 µL/L 1-naphthaleneacetic acid (NAA) and 500 µL/L IBA resulted in significant regeneration of Populus trichocarpa (Nisqually-1) (Abbas et al., 2020).Similarly, other researchers propagated P. trichocarpa on MS medium in order to perform GUS staining and identified expression of PtrCslD5 gene in roots (Peng et al., 2019).Pre-mature embryos of barley (H.vulgare L.) grow very well at 2.5 mg/L concentration of 2,4-D on MS medium and displayed excessive callogenesis, while MS medium enhanced with 1 mg/L Kinetin came out in significant regeneration, and supplementation with 1 mg/L IAA resulted in maximum rooting (Abbas et al., 2023).Callus initiation medium enhanced with MS media and dicamba (2 mg/L) showed maximum efficiency of callus induction of 98.22 and 97.33%, respectively, and shoot development media with addition of zeatin at concentration of 5 mg/L 6-benzylaminopurine (BAP) concentration of 5 mg/L 2,4-D concentration of 0.25 mg/L NAA concentration of 0.25 mg/L, and copper sulfate (CuSO4) concentration of 20 mg/L showed maximum shoot induction in wheat (Phogat et al., 2023).Here, we streamlined MS media enhanced with various concentrations of growth promoting hormones.
Various channels of tissue culture were laid out to upgrade callus formation efficiency in wheat which are extensively including genotypic, culture medium, explant dependent and over expression of callus regenerative gene (Roesler et al., 2018;Chaimae et al., 2021;Yu et al., 2023).For all cultivars, new plants, or species using more than one hormone, tissue culture and growth procedures should be investigated through careful design and testing experiments.Therefore, this study aims to find the most responsive genotypes and most important individuals of wheat having high callus formation and regrowth efficiency, and additionally accelerate the propagation of wheat genotypes that exhibit superior characteristics for genetic advancement based on limited resources and time.The point of our research was to decide reasonable protocol and the selection of most efficient wheat genotype for tissue culture.

Experimental sites and explant selection
In order to establish tissue culture and regeneration in wheat, we selected common, approved and high yield wheat verities being extensively cultivated by farmers.We collected mature seeds of wheat in March 2023, packed in clean and microbe free plastic bags, labeled and stored at normal temperature for further use in experiment.Notably, no specific permission was required to collect samples for experimental purpose.Mature seeds of genotypically different six wheat varieties namely Chakwal-50, Galaxy-01, NARC-09, Pakistan-13, Millet-11 and Borlog-14 were employed for callogenesis and regeneration.Laboratory conditions were adjusted as follows: 25±2°C temperature, 60-80% humidity, and white light Table 1.MS medium enhanced with dissimilar concentrations of IAA and Kinetin for regeneration, while 2,4-D for callogenesis.

Surface sterilization and inoculation of seeds
Mature seeds of all six wheat varieties were picked out as an explant material and disinfected three times with 50% Clorox for 20 min with constant agitation in a laminar airflow hood.Washing of seeds were performed thrice for 10 min with ddH 2 O to remove Clorox and placed at sterilized filter paper to become dry.Disinfected seeds were then shifted on an autoclaved Whatmann filter papers to absorb excessive water.After that, disinfected seeds were cultured on MS media supplemented with regeneration and callus initiation media with the help of sanitized forceps.All experimental procedure was performed inside 6-inch vicinity of spirit lamp.

Regeneration and callogenesis
In order to initiate regeneration, surface sterilized dry seeds of wheat were cultured on MS media supplemented with different concentrations of IAA and Kinetin (Table 1).Each variety of wheat was tested at six different combinations of hormones in order to observe their regeneration potential, and response to various combinations of hormones, on the base of different genetic makeup.Each treatment was performed thrice, data was collected, and average was calculated to avoid any error.Inoculated seeds were incubated in growth room at temperature at 25 ± 1°C, 60-80% humidity, and 7000 lux intense white light.Germination initiated after 4 days of culturing and day-by-day germination data was collected and saved on excel sheet.For the purpose of callus production, inoculation of surface sterilized seeds was performed on MS media enhanced with various levels of 2,4-D (Table 1), and cultures were placed in dark condition for prompt callus induction.Notably, after 2 weeks of incubation in dark condition significant callus formation was observed in almost all wheat verities.All reading were obtained in next 3 to 4 weeks, then separation of calli from seeds was performed with the help of sterilized forceps under sterilized conditions in order to identify best wheat variety with maximum potential of callogenesis.

Statistical analysis
Collected data were statistically analyzed by using slide writer software and ANOVA to check out the differences between treatments and within the treatments, in concern of percentage of callus formation and tissues regeneration.To get optimum result, 20 explants of every variety used for each treatment in three replications.The mean was processed from each treatment.

Study aim
The aim of this study was to foster a strategy for compelling crop improvement via genetic modification.For this purpose, an established and reproducible callus induction and regeneration protocol was optimized using mature seeds of six genetically different wheat genotypes.Although, fully mature seeds of wheat are available around the year, but we collected seeds of six different verities of wheat in March 2023, labelled name of each wheat variety, and stored in sterile bags in order to avoid exposure to microbes such as fungal spores.Notably, it is very hard to induce callus formation in mature seeds as compared to immature embryo, cotyledons or leaves, but mature seeds are available around the year.This is why we employed mature seeds for callogenesis and regeneration.This optimized callus induction and regeneration protocol will be further employed in development of biofortified wheat verities with biotic and abiotic stress resistance.For the purpose to check the potency of six wheat cultivars for callus development and regeneration, six unique combinations of hormones (IAA, Kinetin and 2,4-D) for six treatments were applied.

Regeneration
Diverse concentrations and combinations of IAA (1.5, 3, and 4.5 mg/L) and Kinetin (1, 2, 3, and 4 mg/L) were considered to advance the regeneration combination (Table 1).To obtain high regeneration activity, various amount of Kinetin was mixed in MS media enhanced with multiple levels of IAA.The outcomes showed significant contrast among wheat varieties against growth regulators (IAA and Kinetin) of various levels.
Regeneration of tissues is genotypes dependent; therefore, behavior of different wheat genotypes was different at various concentrations of growth regulators.Millet-11 showed the highest regeneration potency on IAA concentration of 1.5 mg/L and on Kinetin concentration of 5 mg/L on tissue regeneration media (Figure 1).Behavior and regeneration ability of each genotype altered with alteration of IAA and Kinetin levels (Figure 2).However, maximum average regeneration among varieties at all combination of growth regulators was examined in Millet-11 (31.24%), next to 29.16% in Borlog-14, and 27.7% mean maximum regeneration was acquired among all varieties at combination of IAA and Kinetin 1.5 + 2 mg/L, respectively.Wheat cultivar NARC-09 was considered a poor genotype in terms of regeneration as it produced minimum regeneration percentage of 16.6% declared as least active genotype to  among all wheat cultivars for the purpose of tissue culturing (Table 2).However, outcomes of this research presented Millet-11 cultivars as best wheat cultivars for use of tissue culture on the base of giving maximum percentage of regeneration and Borlog-14 was considered as second-best genotypes having potency of regeneration.

Callus induction
Callus formation is the essential step in different processes involved in applied and analytical tissue culture.The callus formation was divided into two different calli.Calli that were dense in look, nodules like shape, creamy-whitish to light greenish in color and embryo-like structures were named as "embryogenic calli" (Figure 4a).Calli that showed color of dirty-whit and soft-watery texture were named as "non-embryogenic calli" (Figure 4b).For callus induction, hormones play important role and, in our research, we found that only 2,4-D was prime for callus development and it was elaborated that 2,4-D is the mostly utilized growth regulators for the purpose of callus formation and its maintenance (Aadel et al., 2016;Eshagi et al., 2021;Phogat et al., 2023).
For the purpose of callus development, various concentrations of hormone 2,4-D (0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 mg/L) were evaluated.In order to find out excellent wheat genotype in terms of callus induction, six wheat genotypes were treated with six different levels of 2,4-D.Callus formation examined as a white spongy tissue on upper side of seed during the periods of 10 to 12 days and it was also genotype and medium dependent (Figure 3).Almost same types of results were disclosed by Mehmood et al. (2013).Culturing was carried out in 2,4-D hormones for up to three weeks and after that data was calculated." The data indicated that behavior of each variety was significantly different under unchangeable cultural environment and medium concentration because of their potency to develop callus.However, callus induction media ought to be well standardized to study the potency of a genotype to make maximum callus on the base of its genetic makeup.Results showed that in the light of different levels of 2,4-D, Millet-11 (41.66%) recovered maximum number of calli percentage at 2,4-D concentration of 2.5 mg/L next to 33.33% by variety Chakwal-50 and 29.16% by variety Borlog-14 (Figure 5).
Other than this, variety Chakwal-50 gave the best a b  outcomes by producing maximum callus weight (0.26 g) at MS media holding 3.5 mg/L 2,4-D followed by Borlog-14 (0.16 g) and Millet-11 (0.14 g) (Figure 6).In general examination of multiple varieties at every giving level of 2,4-D media disclosed that Millet-11 (26.39%) is a genotype exhibited superior characteristics for callus development and 2.5 mg/L concentration of 2,4-D was viewed as best concentration for production of maximum callus (Table 3), and 3.5 mg/L 2,4-D was viewed best for wheat crop to produce highest weight of callus (Table 4).

DISCUSSION
Tissue culture is one of the latest techniques that involves exposure of plant tissue to a specific type of nutrients, different kinds of hormones and artificial light under invitro circumstances to develop many plants, all of them are clones of explants, over a very small interval of time.
Plant tissue culture is a crucial technique for making disease-free, stress resistant planting material and in short time production of uniform plants in developing countries.In botany, callus cells are that kinds of cells that heal plant wound after injury.Callus formation is stimulated from any type of explants materials after being sterilized and in-vitro planting on culture medium contains different hormones and growth regulators.Plants growth controlling hormones such as IAA, Kinetin, and 2,4-D are added in plating medium for the purpose to commence callus production or somatic embryogenesis.
In the present research, six wheat genotypes were utilized to observed in-vitro effect of different hormones  by applying different growth regulators.The role of IAA alone for plant regeneration was not effective but combined effect of IAA and BAP together was vital in case of whole plant regeneration.These findings are consistent with Malik et al. (2004), they elaborated the combined effect of IAA and BAP on whole plant regeneration is necessary as compared to apply these hormones separately on wheat genotypes and found regeneration of plant step-up 84 and 52% in wheat cultivars Inqilab-91 and Pavon-76, respectively.Pattern of callus development and tissue regeneration was varied in all studied genotypes by altering the IAA and Kinetin concentration.However, ability and efficiency of Millet-11 for callus development and plant regeneration was highest in comparison to other genotypes, maybe because of high expression of callus formation and plant regeneration genes at genetics level.Highest amount of callus production was obtained at 2.5 mg/L 2,4-D concentration (Figure 5) while highest callus weight was generated at 3.5 mg/L 2,4-D concentration in wheat (Figure 6).High weight of callus is necessary to establish a good protocol for gene transformation and fast development of plantlets through tissue culture.By the way, the output of our current experiments for callus formation and plant regeneration are consistent with other researchers who also applied different combinations of hormones to establish a tissue culture protocol in wheat.For example, Malik et al. (2021) obtained the most  2021) recorded the highest proliferation and callus induction at concentration of 2 mg/L 2,4-D, in wheat, while highest plants regeneration was observed at concentration of IAA 6 mg/L and 6 mg/L Kinetin.Afzal et al. (2010) mentioned the highest callus formation at 3 mg/L of 2,4-D.Miroshnichenko et al. (2017) observed 10 shoots per explant in wheat by applying 3 mg/L Dicamba concentrations, 50 mg/L concertation of Daminozide, and with TZD concentration of 0.25 mg/L.Callus formation activity of wheat varieties to 2,4-D explained that genetic makeup of each variety have different demands of 2,4-D for maximal callus formation.Different cultivars of wheat have different behavior toward various amounts of plant growth hormones.Callogenesis and regeneration of varieties and variation among them were found to be genotype-dependent (Kagami et al., 2016;Ahmad et al., 2021).

Conclusion
Optimize concentration of the plant growth controlling hormones is crucial for maximal plantlets regrowth for most researches involving tissue development techniques.Among six researched wheat cultivars, high potential for tissue regeneration and callus development was observed by the variety Millet-11.The results also suggest that regeneration potency of a genotype can speed up to a particular level by combining 1.5 mg/L concentration of IAA and 2 mg/L Kinetin concentration.Similarly, 2,4-D is a necessary plant growth hormones and appropriate amount is crucial for different plant species for callus formation and in wheat, 3.5 mg/L 2,4-D concertation was described best for high weight callus induction, so these results might be useful in future for gene transformation for the improvement of agronomic traits.Maximum potential for tissue culture of Millet-11 suggests the opportunity to use it for wheat improvement programs.These findings provide precise knowledge for plant breeders to develop new potential varieties of wheat having strong biotic and abiotic stress resistance in order to get maximum yield to feed growing population.

Figure 3 .
Figure 3. Initiation of callus started as a white spongy tissue.

Figure 5 .
Figure 5. Callus formation percentage of various wheat varieties in response to multiple 2,4-D concentrations.

Figure 6 .
Figure 6.Callus weight of various wheat varieties in response to multiple 2,4-D concentrations

Table 2 .
Effect of IAA and KN on regeneration (%) of wheat.