Control of hyperhydricity of Pistacia khinjuk stocks in vitro shoots (2024)

Plant material and culture condition

P. khinjuk seeds were obtained from Batman Provincial Directorate of Agriculture and Forestry in November 2022. P. khinjuk trees were identified by Turkish Plants Data Service [16] in west of Raman Mountain (37°49’23”N, 41^°08’01”E) Batman province in Türkiye. The fruits and coats were removed from the seeds to obtain kernels. The kernels were disinfected with 20% commercial sodium hypochloride for 20min on a shaker at 200rpm. Then, the seeds were washed three times with sterile distilled water before being placed in contact with 50ml MS [17] in Magenta vessels (GA-7, Sigma Ltd.). The seed germination medium contains 6.5g/L agar and without plant growth regulators and sucrose. Shoot tips or nodal bud segments obtained from the germinated seeds were proliferated in order to obtain stock cultures in standard MS medium containing 1mg/L benzylaminopurine (BAP). All explants (shoot tips or nodal bud) that were used for the experimnts approxymately 1cm long. All medium that were used for the determination of any parameter containing 30g/L sucrose and 6.5g/L agar and plant growth regulators. The pH of all medium was adjusted to 5.8 before autoclaving (120°C for 20min.). Plant growth regulators were added to the medium prior to adjustment of pH and sterilization. Cultures were incubated at 25 ± 2°C under 16/8h (Light/Dark) photoperiods with light conditions of 3500lx created by white fuorescent light for 28 days. All experiments were repeated two or three times.

Calculation of hyperhydricity rate, average length of shoots, proliferation rate, average number of shoots and water content, analyses of soluble total protein, chlorophyll a, b and carotenoid were done instantly after incubation.

Standard MS medium was modified by different concentrations (Table1) of NH₄NO₃ (1650, 825, 412.5 and 206.25mg/L) and CaCl₂·2H₂O (440, 220, 110 and 55mg/L). These concentrations were combined with each other.

Determination of chlorophyll and carotenoid

The chlorophyll (chl) and carotenoid contents were extracted using 80% acetone from the fresh micropropagated leaves of P. khinjuk. The absorbance of the extracts was measured with a spectrophotometer at 480, 663, and 645nm. Chlorophyll a, chlorophyll b, and carotenoid contents were calculated with the following equations [18]:

Chlorophyll a: 12.7(A663) – 2.69(A645)

Chlorophyll b: 22.9(A645) – 4.68(A663)

Carotenoid: [A480 +(0.114(A663) – (0.638-A645)]×V/1000×W

Determination of soluble protein

To determine total soluble protein content, 1g of fresh leaf samples was homogenized with a chilled pestle and mortar in 5 mL of extraction buffer (0.1M phosphate buffer, pH 7.0), containing 10 mM KCl, 1 mM MgCl2, 10 mM Na₂EDTA, and 1% polyvinyl poly pyrrolidone (PVPP), and centrifuged. Next, the supernatant phase was sampled to determine protein content, based on a standard curve prepared with Bovine Serum Albumin (BSA) and expressed as µg g-1 fresh weight [19]. From the fresh plant sample, 0.5g was homogenized in a 100 mM phosphate buffer (pH 7.0) and centrifuged at + 4°C and 17,530g for 20min. 20µl of the supernatant, 480µl distilled water, and 5000µl Bradford solution were added and the absorbance was measured with a UV-Vis spectrophotometer at a wavelength of 595nm.

Determination of shoots fresh weight

Twenty shoots were harvested and immediately weighed on a digital scale with an accuracy of 0.001g.

Determination of shoots dry weight

Twenty explant shoots were placed in a paper bag and dried in an oven at 70°C for 72h and then weighed.

Determination of water content of explant (%)

Water content of explant (%): Fresh weight – dry weight / fresh weight × 100.

Determination of hyperhydric shoots

The shoots that have fragile, translucent stems and curled leaves have been accepted as hyperhydric shoots.

Determination of hyperhydric shoots rate

Hyperhydric shoots/normal shoots x 100.

Determination of proliferation rate

Apical and lateral buds that produce new shoots/ Apical and lateral buds that don’t produce new shoots x100.

Data replication and statistical analysis

The data were assessed by analysis of variance (ANOVA) and the signifcant diferences among mean values (P ≤ 0.05) were evaluated by Duncan’s multiple range test (DMRT).

Effect of NH₄NO₃ and CaCl₂·2H₂O on hyperhydricity

Table1 was noted that reducing concentrations of NH₄NO₃ from 1650 to 206.25mg/L were resulted in an increase of hyperhydric shoots rate. Symptom of hyperhydric shoots grown with lower concentrations of NH₄NO₃ had curled leaves; those grown with higher concentrations of CaCl₂·2H₂O had wrinkled leaves and pale green stems (Fig.1). Table1 shows that the lowest rate of hyperhydric shoots (34.30%) was grown in the medium containing1650 mg/L NH₄NO₃ and 110mg/L CaCl₂·2H₂O. The highest rate of hyperhydric shoots (68.42%) was grown in the medium containing 206.25mg/L NH₄NO₃ and 440mg/L CaCl₂·2H₂O.

Effect of BAP and mT on hyperhydricity

Table1 shows that mT is far more effective than BAP to reduce hyperhydricity rate. The hyperhydricity rate of mT medium were lower than in BAP medium in the all experiments. In addition, reducing concentration of CaCl₂·2H₂O from 440 (standard MS medium level) to 110 (modified MS medium level) mg/L can be say a synergistic effect on shoot performance. Since, shoot performance results of 110mg/L CaCl₂·2H₂O medium is higher than 440 CaCl₂·2H₂O medium according to the number of shoots per explant, average shoot length and proliferation rate in same type and level of plant growth regulatoars medium (Table2). The highest value shoots of average number shoots of per explant (2.45), average shoots length (18.47mm) and proliferation rate (85%) were obtained from the medium containing 110mg/L CaCl₂·2H₂O (Table2). Shoots characteristics of BAP medium had mostly semitransparent, thickened and short internodes like Fig.1B. On the other hand, shoots characteristics of mT medium had mostly turgid and brittle leaves like Fig.1D. The highest rate of hyperhydric shoots (68.42%) was obtained from the MS medium containing 206.25mg/L NH₄NO₃, 440mg/L CaCl₂·2H₂O and 0.5mg/L BAP. The lowest rate of hyperhydric shoots (34.30%) was obtained from the MS media containing 1650mg/L NH₄NO₃, 110mg/L CaCl₂·2H₂O and 1mg/L mT.

Effect of the combinations of NH₄NO₃ and CaCl₂·₂H₂O on shoot propagation

Shoot propagation occurred in all treatments tested. Standard MS medium consisted of the lowest BAP (0.5mg/L) was produced pale green leaves and shoots stem. Vigorous and high quality shoot was produced on modified MS medium that supplemented with 1mg/L mT. Increasing the concentration of mT from 0.5 to 1mg/L, resulted in an increase the average length of shoot from 17.38 to18.47, average number of shoot from 2.30 to 2.45 and proliferation rate from 80.30 to 85.74 but only on modified medium with mT (Table2). When the results of the number of shoots per explant, average shoot length, and proliferation rate in modified MS medium (1650mg/L NH₄NO₃ and 110mg/L CaCl₂·2H₂O) that caused the lowest hyperhydricity rate and standard MS nutrient medium (1650mg/L NH₄NO₃ and 440mg/L CaCl₂·2H₂O) were compared, the most shoots per explant (2.45), longest average shoot length (18.47mm) and highest proliferation rate (85%) have been observed in modified MS nutrient medium containing 1mg/L mT (Table2).

The analysis of soluble protein

Soluble protein content of normal shoots was higher (2.12 ± 0.38) than hyperhydric shoots (1.66 ± 0.28) (Table3).

The analysis of chlorophyll

Chlorophyll a content of normal shoots (0.61 ± 0.56) was higher than hyperhydric shoots (0.43 ± 0.18) and chlorophyll b content of normal shoots (0.35 ± 0.26) was higher than hyperhydric shoots (0.25 ± 0.35) (Table3). Hyperhydric shoots were appeared pale green and translucent than normal shoots.

The analysis of carotenoid

Carotenoid content of hyperhydric shoots (18.29 ± 1.25) were higher than normal shoots (11.75 ± 0.48) (Table3).

The analysis of water percentage

Water percentage of hyperhydric shoots (88.55) were higher than normal shoots (78.70) (Table3). Shoots of high water content had very fragile and thickened stem and leaves.

Recent studies show that the content of NH₄NO₃ in the medium affects the level of hyperhydric tissues of in vitro propagated plants [20]. Such as, Jan et al. [21] reported that high levels of NH₄NO₃ and KNO₃ caused a dramatic increase in hyperhydricity rate of Salvia santolinifolia. Also in some study, lower concentrations of NH₄NO₃ reduce the level of hyperhydricity in different species [22] and hyperhydricity was not observed in MS medium without NH₄NO₃ on in vitro shoot of Brassica oleracea [23]. A decrease (2–3 times) in the concentration of NH₄NO₃ in MS medium reduces the level of hyperhydricity in Prunus avium [24] and Phoenix dactylifera [24]. Hyperhydricity decrease in plum when using medium with a low NH₄NO₃ and KNO₃ content of MS medium [24]. This study could show a causal link between hyperhydricity and NH₄NO₃ level. Since the result of this study confirms recent studies and suggest that an increase in the level of NH₄NO₃ from 206.25 to 1650mg/L can be reduce hyperhydricity rate of micropropagated P. khinjuk shoots.

The correlate of the degree of hyperhydric tissue rate on the high concentration of CaCl₂·2H₂O in the medium was observed in in vitro cultivated shoots of apple and gerbera [25], it has been shown that the presence of CaCl₂·2H₂O in the medium at high concentrations can cause hyperhydricity. Jan et al. [20] reported that high levels of CaCl₂·2H₂O in the medium caused a dramatic increase in hyperhydricity rate of in vitro Salvia santolinifolia shoots. In addition, Machado et al. [26] reported that high concentrations of CaCl₂·2H₂O reduced hyperhydric tissue rate of Lavandula angustifolia shoots. Increasing CaCl₂·2H₂O concentration is an effective means of reducing hyperhydric tissues in some tree species too [24]. P. khinjuk shoots that grown in this study, were grown in the different level of CaCl₂·2H₂O medium had not a certain link between hyperhydricity and level of CaCl₂·2H₂O. Since significant difference was not observed in hyperhydricity rate of CaCl₂·2H₂O concentration from 110 to 440mg/L. So we could not show a certain corelation between hyperhydricity rate and CaCl₂·2H₂O level in in vitro P. khinjuk shoots.

Recent studies have been shown that the presence of benzyladenine (BA) in the medium at both low and high concentrations can cause hyperhydricity for carnation, apple and pine [27]. High concentrations of BA induced high hyperhydricity rate of in vitro Salvia santolinifolia shoots on standard MS medium [21]. Furthermore, Jan et al. [21] discovered that using BA produced higher percentage of hyperhydricity than 2iP in same study. Also, Liu et al. [28] observed that rate of hyperhydricity of in vitro Allium sativum shoots was higher with kinetin than BA. Similarly, Abdouli et al. [15] reported that mT was more effective than BA in reducing hyperhydricity rate in in vitro Pictacia vera shoots. Replacement of BA with mT also led to a decrease in hyperhydricity symptoms in apple and pear shoots [29]. This study shows that increase the concentration of BAP and mT from 0.5 to 1mg/L were reduced the hyperhydricity rate of in vitro shoots of P. khinjuk on modified and non modified MS medium, but mT was far more effective according to low hyperhydricity rate. This study agrees with previous studies that the type and the density of phytohormone affect the hyperhydricity of in vitro shoots.

Type of explants, culture medium, variety and density of growth regulators, humidity [30], type of gelrite and sucrose density were caused stress on in vitro explants [31]. The function of carotenoids is associated with plant response to environmental stresses [32]. Light, temperature, chilling, drought, and salinity change carotenoid content [24]. Recent studies have indicated that stress factors in culture medium could result in hyperhydric tissue for garlic, sugar beet [24, 26], and Dendrobium officinale [33]. This study results showed that the carotenoid content of hyperhydric shoots was significantly higher in hyperhydric shoots than in normal shoots. This may indicate that hyperhydric shoots genetically are very sensitive than normal shoots. Since particular types of plants and even different genotypes of the same plants can have different responses in same in vitro culture conditions [34].

Hyperhydric tissue has inadequate chlorophyll and surplus fluid in the intercellular spaces. This changes enzyme activity and protein synthesis and normal metabolic processes [12]. Type and concentration of cytokinin were caused the low protein content in hyperhydric explants [20] Recent studies show that hyperhydric explants have lower protein contents than normal explants. The results of this study are similar to recent studies since, hyperhydric shoots were found to have lower soluble protein content than normal shoots.

Hyperhydric tissue has multiple deficiencies such as appear turgid, watery at their surface, contain much water in tissue intercellular spaces, less lignification, somewhat transculent, in some cases less green than normal and brittle [14]. This aspect has been reported for in vitro shoots of gerbera [35], carnation [36], shallot [37], Annona glabra [38], and Dendrobium officinale [34]. Hyperhydric stem and leaves of this study have a pale green appearance. Since chlorophyll a, b content of normal shoots was higher than hyperhydric shoot and water content of hyperhydric shoot was higher than normal shoots.The pale green appearance can orginate from high water and low chlorophyll content of hyperhydric stem and leaves.

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Authors and Affiliations

1. Department of Food Processing, Vocational School of Technical Science, Batman University, Batman, Turkey

   Yusuf Ersali

Contributions

The experimental studies, analysis, writing and review of this manuscript were made by Y. E.

Corresponding author

Correspondence to Yusuf Ersali.

Ethics approval and consent to participate

No ethical approval and consent to participate.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

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