Wheatgrass is a type of prominent crops belongs to poaceae family. Many seasonable  cereals  such  as  wheat,  barley,  oat,  rye  and  triticale are acceptable crops for  in  vitro regeneration. Since a decades, many scientists and researchers involves in regeneration of allied species of crops for producing transgenic plants (Gales et al. 1998). In cereals, the crops are transformed to mature plant with the application of many Biotechnology applications. The use of cell and tissue culture systems has a significant impact to produce disease resistant variety of crops. Mostly cereal  regeneration  includes  induction of  embryogenesis  calluses  from  immature  tissues  and  further subsequent regeneration  of  plantlets (Jimenez et al. 2001). In most of the cases, the process of regeneration is seems to be time-consuming and laborious process.  Many literature surveys on somaclonal variation in different types of crops show multitasking results of callus induction and regeneration process (Bennid et al. 1996). In some cases, the abnormal morphological characteristics and reduced fertility were also reported in many cultured crops during Plant tissue Culture Technique using Plant Growth Regulators (Trewavas 1981). In contrast to immature tissue, explants are seems to be most active derived from immature embryos or scutella in wheat in  culture medium[1].  In some reports, the required culture and induction of direct shoot regeneration depends on the nature of the plant organ (Ganeshan et al. 2006)  which the explant was regenerated. It is highly responsive on character of plant genetic modification (Repellin et al. 2001). The direct shoot  proliferation  protocol depend on the nature  of medium  and the  type of  the  explants  for elite  cultivars of  wheat. The activation of shooting response depends on the concentration of cytokinin supplemented in the medium. Cytokinins works as signaling molecules that activate totipotent cells of callus for shoot organogenesis. These molecules activate as perfect machinery of somatic (Eudes et al. 2003) cells (leaf, stem, cotyledon and so on) for direct organogenesis, while of shoot apex they stimulate the growth caused by presence of meristemic cells at the tip of explants. These cytokinin may also shows a positive impact on the explants to produce multiple shooting responses.

Taxonomy

Kingdom             Plantae

Division              Magnoliophyta

Class                   Liliopsida

Order                  Cyperales

Family                 Poaceae

Genus                 Triticum

Species               Aestivum

Common            Wheatgrass

Name

History of Wheatgrass (Triticum aestivum)

Wheatgrass has been used in Indian tradition since a decades. The consumption of wheatgrass was first began in western world in the 1930.  Later, it spread throughout the western countries like United States and Canada. Dr. Charles F. Schnalad first highlighted the role of the use of Wheatgrass in our diet in 1932. Besides, he also developed a product called “Carophyl” which is known as “World First Multivitamin” (Figure 1d). The glass of wheatgrass in our daily diet also provides beneficial aspects for human health. Besides, multivitamin tablets also have a good impact which provides all vitamins for perfect growth and development of body (Figure 1f & 1g). Since decades, Wheatgrass is traditionally used in both Persian and Indian festivals. Many rituals of Hindu culture are done with cultivating wheatgrass. During Navaratri pooja, Hindu sow wheatgrass seeds on the first day of Navaratri pooja and offer the saplings to the mother goddess on the last day as part of the rituals.

The role of micro-propagation has a good link with regeneration and callus morphogenesis (George et al. 2005). The abiotic factors like temperature, salinity, heat, pressure plays a great role on growth and development for expalnts in even aseptic condition. Light condition during in vitro phase had a positive influence on the responses of explants to direct shoot regeneration (Alizadeh et al. 2004). The explants that initially were kept in darkness and then exposure to photoperiod showed better response than those maintained in photoperiod treatment. That is, darkness was observed to stimulate more direct shoots than light condition. These findings were supported by the studies in and on wheat. The effect of light can be a good source of metabolism and sugar uptake. Many scientist and researcher reported a good response of hormones ( Davis et al. 1995) in  developing protocol for shoot embryonic meristem with the scutellum, the combination of 10 mgl-1 (BAP) and 2 mgl-1 (2, 4-D) (Fujimura et al. 1980) and darkness for 4 weeks can enable to enhance the rate of plant regeneration (Sharma et al. 2004) and do reduce the number of days required to direct shoot regeneration using in vitro culture. 

Literature Survey and Collection of seeds

A regular literature survey was done before collection of seeds. A good variety of seeds was brought from nearby seed bank nearby Madhav University Campus, Aburoad Rajasthan.

Preparation of MS Media

Composition of Murashige and Skoog's (MS)  Media  (Murashige and Skoog 1962)

Germination of Seeds

Here the seeds was first sterilized in CuSo₄ Solution, Later they were allowed to germinate on filter paper moisten with nutrient solution i.e MS media having macronutrient, micronutrient and Vitamin solution for 12 hour. After 12 hour, the seeds spread over the filter paper in four individual petriplates. The petriplates were kept at different parameters like light and dark condition. The temperature was maintained  at 22 to 25⁰C. These petridish were kept in a thermostatically controlled culture room at 25 + 2⁰C under 12 : 12 light : dark cycle.  

Media used

The germinated seeds inoculated in plant growth regulators (Auxin, Gibberlin, Cytokinin and IBA) supplement Agar plus MS Media, The amount of Plant Growth Regulators was 10^-3M, 10^-6M and 10^-9M.

In vitro culture of different types of crops

Transfer of In vitro cultured Plants to In vivo condition

The process of  germination started within 48 hours. When the size of the seedlings reached about 5 cm, the plant pot was transferred to the greenhouse. The temperature of the greenhouse was constantly maintained at 25 ± 2⁰C, and 900 lux of artificial cool fluorescent light with a 14 hour photoperiod was provided to compensate the short day length. The height of mature plants ranged from 115cm after 7 days of inoculation inside the soil.

Nutritional and Mineral content was Analysis by AOAC 1990 and Pavel 2009.

Wheatgrass is a major source of nutrients essential for health. It can be cultivated in throughout the year. Wheatgrass can be grown indoors or outdoors. The role of Plant Tissue Culture techniques have a great significant role to fertile the wide verities of crops in short interval of time. In vivo condition, it is not possible to cultivate in all season but in vitro condition, it can grow throughout the year in aseptic condition as we desire.

Figure 1a: A bunch of elongated shoots of Triticum aestivum (Wheatgrass) cultured in MS Nutrient supplement Plant Growth regulator media.

Figure 1b: Regenerated Shoots and Roots of Triticum aestivum (Wheatgrass) in MS plus Plant Growth regulators supplemented solid media.

Figure 1c: Regenerated shoots transfer to pot for further growth of Triticum aestivum (Wheatgrass) plants.

Figure 1d: Juice extractor to prepare the juice of Shoots of Triticum aestivum (Wheatgrass) plants.

Figure 1e: A fresh glass of Wheatgrass Juice.

Figure 1f: A bottle of Wheatgrass tablets.

Figure 1g: A health tonic of Wheatgrass called Carophyl.

In in vitro condition, it grows in MS medium with supplementation of Plant Growth Regulators. Leaves are harvested when they develop a "split" as another leaf emerges. George et al (2008) reported a brilliant view on micro-propagation during Tissue culture.  In most of the literature, the callus phase is exclusive concession that used in Rice (Nhuts et al. 2000), cereal (Eudes et al. 2003) to decrease the time, spend, frequency of somaclonal variation and especially genotype dependency that is consistent to (Sharma et al. 2004). Similar genotype were developed in present study. Here different types of plant growth regulators were taken. It includes Auxin, Gibberlin, Cytokinin and IBA. These hormones were taken in different concentration like 10^-3M, 10^-6M and 10^-9M, MS media was taken which was supplemented with different concentration of hormones. Here two types of media were used i.e liquid and solid media. The liquid media was supplemented with hormones and MS media, while in solid media both MS and 0.8% Nutrient Agar were used. In liquid media, seed culture was done in Ms media supplemented with hormones. Out of four types of Plant Growth Regulators, Auxin plays a good role in morphogenesis and growth of shoots and roots in both liquid and solid culture. The activation of shooting response depends on the concentration of cytokinin supplemented in the solid medium (Figure 1b). Cytokinin also shows a positive attitude of signaling molecules that activate totipotent cells of callus for shoot organogenesis. These molecules activate somatic cells (Shoots, roots and cotyledon) for direct organogenesis ((Malik et al. 2007, Nhut et al. 2002).  The shoots were emerges out and stimulate the growth occur due to meristemic cells at the tip of explants. These cytokinines may also turn the explants to produce multiple shooting responses (Centeno et al. 1996) . At the tip of the plantlets water droplets are observe which shows the sign of Guttation (Figure 1c). Our results reported on the seed germination started within 48 hours of inoculation in 10^-6M Auxin supplemented MS media (Figure 1a and 1c). However, within one week of short interval of period, the shoots grow out measures 17cm in and the length of roots ranges upto 5cm in length. The color of shoots was dark green.  Similarly, The roots were developed elongated with sharp root hairs, dark brown in color. On the other hand, In solid nutrient Agar supplemented plant growth regulator combination, the shoots and roots ranges The meristem  cells has a great achievements in regeneration of  plantlets in direct regeneration (Ahmad et al. 2001). The role of micro-propagation has a good link with regeneration and callus morphogenesis (George et al. 2005).  The abiotic factors like temperature, salinity, heat, pressure plays a great role on growth and development for expalnts in even aseptic condition. Light condition during in vitro phase had a positive influence on the responses of explants to direct shoot regeneration (Alizadeh et al. 2004). The explants that initially were kept in darkness and then exposure to photoperiod showed better response than those maintained in photoperiod treatment. That is, darkness was observed to stimulate more direct shoots than light condition. These findings were supported by the studies in and on wheat. The effect of light can be a good source of metabolism and sugar uptake. Many scientist and researcher reported a good response of hormones ( Davis et al. 1995) in  developing protocol for shoot embryonic meristem with the scutellum, the combination of 10 mgl-1 (BAP) and 2 mgl-1 (2, 4-D) (Fujimura et al. 1980) and darkness for 4 weeks can enable to enhance the rate of plant regeneration (Sharma et al. 2004) and do reduce the number of days required to direct shoot regeneration using in vitro culture. Similar, response was observed in present study with the combination of Auxin, Gibberlin, Cytokinin and IAA plus MS Agar medium, lastly it has been reported that none of the regenerated plantlets in this study showed morphological abnormalities and set seeds normally. The leaves were erect except for the oldest two or three leaves. The tillers of the five 17 plants continuously supplied panicles at different developmental stages for this experiment. The panicles not used for the experiment developed to maturity. The number of florets on a panicle was estimated to be about 60 to 110. From randomly selected panicles, the rate of sterility was determined to be 84 percent for the earlier panicles. Seed sterility was significantly decreased as subsequent tillers matured to produce their panicles. The growing season and conditions and/or lack of wind to aid self-pollination in the greenhouse was most likely responsible for the high sterility of the plants. The seeds observed from this experiment was good and healthy.

Nutritional and Mineral content

Triticum aestivum is commonly known as Wheatgrass. It is a major stable food in everyone diet. It has rich nutritional components. In comparison to other land plants, wheatgrass also contain a high amount of chlorophyll a, b , c , antioxidant and enzymes. Many researcher and scientist reported the health benefits of wheatgrass in human life. It provides a major source of supplemented nutrition. It is a source of minerals like potassium, calcium, copper, iron, zinc, copper, manganese, selenium etc. Besides, Wheatgrass also contain protein, dietary fiber, vitamin A, vitamin C, vitamin E, vitamin K, Vitamin B, Vitamin B2, Vitamin 6 etc. In contrast, It is also a good source of protein . Khan et al. (1984) has discuss the role of nutritional components of wheatgrass. In his report, positive attribute of nutritional components was engulfed and prescribed to add the wheatgrass products in everyday life. (Table 1). Figure 2 shown a very sharp amount of mineral and other nutritional contents present in wheatgrass. The composition of nutritional component seems to fluctuate. The juice of wheatgrass provides all nutrients necessary for body building biological activity. The nutrient content of wheatgrass juice is almost roughly equivalent to that of dark leafy vegetables. Pavel et al. 2009 reported the techniques on analysis of chemical and nutritional components of different types of crops. Many studies proved the wheatgrass to have freshly sprouted first leaves. They are used as a food, health drink, or dietary supplement.  The juice of wheatgrass juice has a best complements to have a best ingredients in all juice bars to fulfill the body fitness. Due to its high content of bioactive compounds, it could be recommended for consumption as a fresh juice blend to elevate fertility and promote youthfulness. This juice meets the changes in the present-day consumers’ diet chart. It becomes a vital change in the marketing trends of the food sector. It also highly recommended the consumption of juices because of the importance for micronutrient and macronutrient (Figure 1d & 1e). Wheatgrass is available easily in most part of the countries as fresh as produce, in tablets, frozen juice, shakes with fruits, mints, coriander and powder. Besides above, Wheatgrass is also famous in commercial market as a cosmetic, foliar spray, face packs, creams, hair gel, massage lotion, fertilizer and liquid herbal supplement.

Figure2 : Graphical representation of Nutritional and Mineral components of Wheatgrass.

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