**3. The effect of increased water absorption on shoot regeneration**

In another study conducted by Yildiz et al. [4], hypocotyl explants of three flax cultivars ('Omega', 'Fakel' and 'Ariane'), which were pretreated and non-pretreated before culture, were cultured for regeneration. In the study, two regeneration methods, which were based on two

different pretreatment applications, were compared with the conventional regeneration protocol in which explants were directly cultured on MS medium supplemented with 1 mg l−1 BAP and 0.02 mg l −1 NAA. Hypocotyl explants were kept in sterile cabin under air flow for 30 min in order to make them dry as reported by Christmann et al. [18] in the first and second pretreatment applications in order to decrease the tissue water content and to help the tissues gain the ability to uptake increased amount of water, all solutes and plant growth regulators from the growth medium via tissue's higher osmotic pressure. Later, explants were sub‐ merged in MS solution having 1 mg l−1 BAP and 0.02 mg l−1 NAA for 15 min in both pretreat‐ ment applications. Then, explants were cultured on MS medium without growth regulators in the first pretreatment application and on MS medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA in the second pretreatment application. It was thought that drying of explants under air flow in sterile cabin increased tissue's osmotic pressure and enabled all cells to absorb more growth regulators along with water in both pretreatment applications by immersing explants into liquid. On the other hand, explants were cultured on MS medium containing 1 mg l−1 BAP and 0.02 mg l −1 NAA only in the second pretreatment application that means tissues main‐ taineduptaking water andgrowthregulators fromthemediumandthis ledto thehigherresults in all parameters studied as noted by Yildiz and Ozgen [3]. Okubo et al. [19] has reported that regeneration capacity was affected by endogenous hormone levels of tissue significantly. Fatima et al. [20] has also reported that plant growth is affected by the internal factors such as chemicals and mineral nutrients. Endogenous levels of growth regulators of the plant tissue determine the amount of exogenous plant growth regulators required for regeneration [20]. It was firstly reported that keeping the explants in sterile distilled waterfor a while before culture initiation promoted the regeneration capacity of explants by increasing tissue's water content and enabling water, all solutes and growth regulators to transfer into the tissue more easily [3].

In accordance with the results, there were statistically important differences among pretreated and non-pretreated hypocotyls in all cultivars. The highest results in all parameters studied were recorded from the second pretreatment application. On the other hand, the lowest results were obtained from the first pretreatment application in which explants were cultured on MS medium without growth regulators in all cultivars after submerging them in MS solution having 1 mg l−1 BAP and 0.02 mg l−1 NAA for 15 min (**Figure 4**).

Higher results in the fresh and dry weights could be attributed to higher metabolic activity caused by an increase in the absorption of water and growth regulators from the growth medium. From the results of the second pretreatment application, it might be easily seen that culturing explants on MS medium having 1 mg l−1 BAP and 0.02 mg l−1 NAA after submerg‐ ing them in liquid MS medium having 1 mg l −1 BAP and 0.02 mg l −1 NAA increased the tissue's growth regulators' level leading to the higher fresh and dry weights. In fact, transferring explants on MS0 medium after treating them with liquid MS that has 1 mg l −1 BAP and 0.02 mg l −1 NAA for a moment in the first pretreatment application, growth regulators of tissues did not seem to be sufficient for high scores according to fresh and dry weights. Culturing explants directly on MS medium containing 1 mg l −1 BAP and 0.02 mg l −1 NAA were not enough again in the increasing tissue's growth regulators' content to obtain higher scores in characters examined in the non-pretreatment application. All the explants regener‐

different pretreatment applications, were compared with the conventional regeneration protocol in which explants were directly cultured on MS medium supplemented with 1 mg l−1

30 min in order to make them dry as reported by Christmann et al. [18] in the first and second pretreatment applications in order to decrease the tissue water content and to help the tissues gain the ability to uptake increased amount of water, all solutes and plant growth regulators from the growth medium via tissue's higher osmotic pressure. Later, explants were sub‐ merged in MS solution having 1 mg l−1 BAP and 0.02 mg l−1 NAA for 15 min in both pretreat‐ ment applications. Then, explants were cultured on MS medium without growth regulators in the first pretreatment application and on MS medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA in the second pretreatment application. It was thought that drying of explants under air flow in sterile cabin increased tissue's osmotic pressure and enabled all cells to absorb more growth regulators along with water in both pretreatment applications by immersing explants into liquid. On the other hand, explants were cultured on MS medium containing 1 mg l−1 BAP

taineduptaking water andgrowthregulators fromthemediumandthis ledto thehigherresults in all parameters studied as noted by Yildiz and Ozgen [3]. Okubo et al. [19] has reported that regeneration capacity was affected by endogenous hormone levels of tissue significantly. Fatima et al. [20] has also reported that plant growth is affected by the internal factors such as chemicals and mineral nutrients. Endogenous levels of growth regulators of the plant tissue determine the amount of exogenous plant growth regulators required for regeneration [20]. It was firstly reported that keeping the explants in sterile distilled waterfor a while before culture initiation promoted the regeneration capacity of explants by increasing tissue's water content and enabling water, all solutes and growth regulators to transfer into the tissue more easily [3].

In accordance with the results, there were statistically important differences among pretreated and non-pretreated hypocotyls in all cultivars. The highest results in all parameters studied were recorded from the second pretreatment application. On the other hand, the lowest results were obtained from the first pretreatment application in which explants were cultured on MS medium without growth regulators in all cultivars after submerging them in MS solution

Higher results in the fresh and dry weights could be attributed to higher metabolic activity caused by an increase in the absorption of water and growth regulators from the growth medium. From the results of the second pretreatment application, it might be easily seen that culturing explants on MS medium having 1 mg l−1 BAP and 0.02 mg l−1 NAA after submerg‐

growth regulators' level leading to the higher fresh and dry weights. In fact, transferring

tissues did not seem to be sufficient for high scores according to fresh and dry weights.

were not enough again in the increasing tissue's growth regulators' content to obtain higher scores in characters examined in the non-pretreatment application. All the explants regener‐

explants on MS0 medium after treating them with liquid MS that has 1 mg l

−1 BAP and 0.02 mg l

−1 NAA for a moment in the first pretreatment application, growth regulators of

−1 NAA increased the tissue's

−1 BAP and 0.02 mg l

−1 BAP and

−1 NAA

having 1 mg l−1 BAP and 0.02 mg l−1 NAA for 15 min (**Figure 4**).

Culturing explants directly on MS medium containing 1 mg l

ing them in liquid MS medium having 1 mg l

−1 NAA. Hypocotyl explants were kept in sterile cabin under air flow for

−1 NAA only in the second pretreatment application that means tissues main‐

BAP and 0.02 mg l

6 Water Stress in Plants

and 0.02 mg l

0.02 mg l

**Figure 4.** Tissue culture response of pretreated and non-pretreated hypocotyls of three flax cultivars ('Omega', 'Fakel' and 'Ariane') 6 weeks after culture initiation. Value on each the bar is the mean of three cultivars [4].

ated in the second pretreatment application successfully with the regeneration percentage of 100% (**Figures 4** and **5**).

The highest results in shoot number per hypocotyl and shoot length were obtained from second pretreatment application in all cultivars studied. The highest shoot number per hypocotyl was recorded as 8.97. The highest score related to shoot length was 2.14 cm. Shoot regeneration capacity of hypocotyls increased significantly in second pretreatment application. The explants to which second pretreatment application was carried out were more vital and well-grown and more capable of regeneration (**Figures 5(b)** and **6(b)**). The highest total shoot number per Petri dish was obtained as 278.10 from second pretreatment application. Total shoot number per Petri dish was reported as a good indicator of the success in both shoot regeneration percentage and shoot number per explant [21]. The highest result of the total chlorophyll content was achieved from the second pretreatment application as 347.70 μg/g fresh tissue. Emerson [22] reported that there exists a close relationship between photosynthesis and chlorophyll content. Chlorophyll content of leaf is thought as a sign of photosynthetic capacity of tissues [22–25] playing a critical role in plant growth and development [26] and its amount alters under stress conditions [27–29]. Gireesh [30] has informed that chlorophyll can be used for measuring growth.

**Figure 5.** Shoot regeneration from hypocotyl explants of flax cv. 'Omega' [4]. (a) The first pretreatment application: hy‐ pocotyls dried for 30 min in sterile cabin and then they were imbibed to liquid MS medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA for 15 min, and consequently, cultured on MS medium without growth regulators, (b) the second pretreatment application: hypocotyls got dried by waiting for 30 min in sterile cabin and then were imbibed to liquid MS medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA for 15 min, and finally, cultured on MS medium having 1 mg l−1 BAP and 0.02 mg l−1 NAA and (c) non-pretreatment application: hypocotyl explants got directly cultured on MS medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA.

**Figure 6.** Regenerated shoots of cv. 'Omega' from (a) first pretreatment application, (b) second pretreatment applica‐ tion and (c) non-pretreatment application 6 weeks after culture initiation (bar = 1.0 cm) (original).

In accordance with the results, it might be concluded that the lower levels of all the parameters which were recorded in the first and third pretreatment applications caused from a decreased uptake of water and growth regulators directly from the medium. Higher shoot regeneration has been significantly affected by tissue water content [3]. Keeping explants in liquid medium containing 1 mg l−1 BAP and 0.02 mg l−1 NAA for a while before culture enabled water, all solutes and growth regulators to transfer through the tissue easily, providing all the cells a high regeneration capacity.
