**2. The effect of increasing tissue water content on** *in vitro* **regeneration**

It was reported that tissue water content affected explant's shoot regeneration capacity significantly [3]. Yildiz and Ozgen [3] have conducted a study to evaluate the effect of tissue water content on regeneration capacity of hypocotyl explants of flax (*Linum usitatissimum* L.). In the study, water-treated and non–water-treated hypocotyl explants of three flax cultivars ('Madaras', '1186 Sel.' and 'Clarck') obtained from Northern Crop Science Laboratories, North Dakota, USA, were compared with regards to fresh and dry weights, shoot regeneration percentage, shoot number per explant, shoot length and total shoot number per Petri dish. Sterilized seeds were germinated on a basal medium containing the mineral salts and vitamins of Murashige and Skoog (MS) [9], 3% (w/v) sucrose and 0.7% (w/v) agar. Hypocotyl segments of 5 mm length were excised from 7-day-old seedlings. Some hypocotyls were submerged in sterile distilled water and shook gently for 20 min before they were placed on growth medium for regeneration, while the others were directly cultured on MS medium containing 1 mg l−1 6-benzylaminopurine (BAP) and 0.02 mg l−1 naphthaleneacetic acid (NAA) to regenerate. It is clear according to the results that there were sharp and statistically significant differences in all cultivars between water-treated and non-water-treated tissues related with all the charac‐ ters examined (**Figure 1**).

cy shoot regeneration is one of the major objectives for tissue culture studies that is also a prerequisite for an efficient transformation system and a clonal production of plants with

Plant tissue culture techniques have certain advantages overtraditional propagation methods. Via tissue culture methods, thousands of mature plants having desirable traits such as good flowers, fruits and odor can be produced in a short time; endangered species which cannot propagate in native environment can be cloned easily by vegetative parts; genetically identical plants can be produced with large quantities; genetically modified plants can be regenerated from cultured cells; production of disease-, pest- and pathogen-free plants increase the plant production; andplantshavingseedgerminationandgrowingproblems canbe easilyproduced. Plantgrowthregulatorsasmediacomponentsaffectthe shootregenerationcapacityof explants. Tissue culture studies have tried to determine correct combinations of auxins and cytokinins for high-frequency adventitious shoot regeneration for related genotype. However, determination of optimum levels of auxins and cytokinins in growth medium is not the only way of increasing shoot regeneration capacity. It is reported that regeneration capacity of explant could be increased by adjusting the concentration, temperature and application period of NaOCl solutions used for surface sterilization [1] and manipulating physical microenvironment by altering distances among explants cultured resulted in increased shoot regeneration capacity [2]. Recently, it is noted that water capacity of the tissue affects explant's

Source of life is based on water on the earth. Living is limited in a large proportion of terrestrial ecosystems according to water availability. The water content in an actively growing plant can be as much as 95% of its live weight. Wateris needed in a plant for photosynthesis. Carbon dioxide and oxygen which is required for photosynthesis cannot be used by plant if they are not soluble in water. For this reason, water is the main factor for plant's existence and growth.

The decrease in growth, yield and quality by water stress has been reported in field conditions [6,7]. Plant survival is guaranteed by germination and seedling establishment and they are very important phases of plant life. Germination ratio diminishes with decreasing external water potential and there is a critical value of water potential for each species below which

This chapter is aimed to show the effects of water deficiency in tissue on shoot regeneration capacity of the explants cultured under *in vitro* conditions. Moreover, increasing shoot regeneration frequency of explant by enhancing water content of the tissue is another issue this chapter focused on. All the results given here were based on three research studies.

**2. The effect of increasing tissue water content on** *in vitro* **regeneration**

It was reported that tissue water content affected explant's shoot regeneration capacity significantly [3]. Yildiz and Ozgen [3] have conducted a study to evaluate the effect of tissue

), sugars (glucose and sucrose) and amino acids are

interesting flowers and fruits massively for ornamental aims.

regeneration capacity significantly [3–5].

Mineral ions such as potassium (K+

germination will not occur [8].

dissolved in water.

2 Water Stress in Plants

**Figure 1.** Tissue culture response of water-treated (WT) and non-water-treated (NWT) hypocotyl explants of three flax cultivars ('Madaras', '1186 Sel.' and 'Clarck') 6 weeks after culture initiation on MS medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA. Value on each the bar is the mean of three cultivars [3].

In the study, all explants were regenerated in water treatment application while only 75.56% of explants formed shoots in non-water treatment application. Water-treated explants had the highest fresh and dry weights compared to non-water-treated ones at the end of the culture (**Figure 2(a)** and **(b)**). Shoots grown from water-treated explants were more vital and well grown (**Figure 2(c)**) than the ones recovered from non-water-treated explants (**Figure 2(d)**). The highest shoot number per explant and total shoot number per Petri dish were obtained from the water-treated hypocotyl explants as 11.4 and 170.96, respectively. On the other hand, non-water-treated explants gave rise to only 7.14 shoots per explant and 107 shoots totally per Petri dish (**Figure 1**).

**Figure 2.** *In vitro* shoot regeneration in water-treated (a) and non-water-treated (b) hypocotyl explants of cv. '1886 Sel.'. *in vitro* root formation and plantlet development of shoots regenerated from water-treated (c) and non-water-treated (d) explants of cv. '1886 Sel.' [3].

**Figure 3.** *In vitro* root development of shoots regenerated from water-treated (WT) and non-water-treated (NWT) hy‐ pocotyl explants of three flax cultivars ('Madaras', '1186 Sel.' and 'Clarck') on rooting medium enriched with 3 mg l−1 IBA 3 weeks after culture initiation. Value on each the bar is the mean of three cultivars [3].

Shoots got rooted on MS medium supplemented with indole-3-butyric acid (IBA) at a concen‐ tration of 3 mg l−1 for 3 weeks. The highest figures were recorded in the shoots regenerated from water-treated tissues (**Figures 2(c)** and **3**).

In the study, all explants were regenerated in water treatment application while only 75.56% of explants formed shoots in non-water treatment application. Water-treated explants had the highest fresh and dry weights compared to non-water-treated ones at the end of the culture (**Figure 2(a)** and **(b)**). Shoots grown from water-treated explants were more vital and well grown (**Figure 2(c)**) than the ones recovered from non-water-treated explants (**Figure 2(d)**). The highest shoot number per explant and total shoot number per Petri dish were obtained from the water-treated hypocotyl explants as 11.4 and 170.96, respectively. On the other hand, non-water-treated explants gave rise to only 7.14 shoots per explant and 107 shoots totally per

**Figure 2.** *In vitro* shoot regeneration in water-treated (a) and non-water-treated (b) hypocotyl explants of cv. '1886 Sel.'. *in vitro* root formation and plantlet development of shoots regenerated from water-treated (c) and non-water-treated

**Figure 3.** *In vitro* root development of shoots regenerated from water-treated (WT) and non-water-treated (NWT) hy‐ pocotyl explants of three flax cultivars ('Madaras', '1186 Sel.' and 'Clarck') on rooting medium enriched with 3 mg l−1

IBA 3 weeks after culture initiation. Value on each the bar is the mean of three cultivars [3].

Petri dish (**Figure 1**).

4 Water Stress in Plants

(d) explants of cv. '1886 Sel.' [3].

Statistically significant differences were observed in all parameters between the shoots which were regenerated from water-treated and non–water-treated explants. This sort of effects in water treatment got also noted in the rooting stage. It means that shoots which were regener‐ ated from water-treated explants got more capable of establishing new plantlets than the ones which were grown from non–water-treated explants.

It could be concluded that the lower levels of all parameters of non–water-treated explants were directly due to a decreasing amount of water uptake from the environment and conse‐ quently, a reduced mobilization of plant growth regulators. Application of water treatment to explants before culture initiation enriched the tissue's water content and so enabled all solutes and plant growth regulators to transfer into the tissue, providing all cells with a high regen‐ eration capacity and consequently, increasing explant's tissue culture response. Increased growth in water-treated explants was confirmed by Naylor's [10] study which stated that plant growth regulators promote cell division and cell elongation. It has also been reported that decreased germination and seedling growth in stressed rice seedlings was due to decreased mobilization of starch and α-amylase activity [11].

It is understood that pretreatment of explants with water before culture initiation increased the permeability of the epidermis layer and caused to high metabolic activity by increased uptake of water and hormone from the growth medium. Higher fresh and dry weights of water-treated hypocotyls at the end of culture could be attributed to an increase in the absorption of water and other components from the growth medium by means of high permeable epidermis membrane. Water-treated tissues were observed bigger in size than non– water-treated ones in all cultivars as reported by Dale [12], who pointed out that the fresh weight increase causes the cell enlargement with water absorption, cell vacuolation and turgordriven wall expansion in this study. The increase in dry weight got closely related to cell division and new material synthesis [13]. Dry weight increase of water-treated tissues is caused by an increase in carbohydrate metabolism resulting from the increased water uptake. Besides, lower levels of all the parameters of non-water-treated tissues caused directly a decreased water uptake through the environment and nevertheless, a decreased mobilization of plant growth regulators. Inhibition of the cell division, elongation of cell, or both of them led to the inhibition of growth under water stress conditions [14]. Cell elongation is affected by osmotic water absorption. Osmotic stress lead to biochemical changes in cell wall during growth [15]. Osmotic stress inhibits water uptake which is vital for germination and growth [16]. And water stress affects the level of plant hormones significantly [17].
