In Vitro Prevention of Browning in Persian Walnut ( Juglans regia L.) cv. Sulaiman

: The present investigation was undertaken to standardize the media and the anti-browning regime in order to minimize the phenolic browning of an in vitro culture of Persian walnut cv. Sulaiman. The experiments involved two types of explants, forced and unforced shoot tips, two types of media, Driver and Kuniyuki Walnut (DKW) medium andMurashige and Skoo ‘ gs (MS) medium, and three types of anti-browning agents, namely, Polyvinylpyrrolidone, ascorbic acid and activated charcoal at 150, 350 and 550 mg/L each. The investigation was replicated thrice under a completely randomized design. Forced shoot tips of cv. Sulaiman on DKW medium showed the best performance in terms of least browning (13.6 ± 10.5%) and highest survival percentage of explants (74.5 ± 2.4%) when treated with ascorbic acid at 550 mg/L. However, unforced shoot tips in MS medium did not perform well and manifested maximum browning (52.9 ± 5.2%). Based on the results, we conclude that incorporation of ascorbic acid in the DKW medium signiﬁcantly reduced the media and explant browning, thus, it could set the basis of successful in vitro-propagation of walnuts.


Introduction
The Persian walnut (Juglans regia L., family: juglandaceae) is one of the important species of the genus Juglans. It originated in Central Asia and its wild and semi-cultivated ancestors have been found in the Balkans across Turkey, China and Eastern Himalaya [1][2][3]. Later, it spread to Western Asia and Eastern Europe from Xinjiang province of China, parts of Kazakistan, Uzbakistan, Kyrgistan, and from Iran and the Central Himalayan region [4,5].
The walnut tree is a prominent temperate nut crop having great potential for the production of nuts and is rich in nutrients and mineral matter [6]. Nuts are abundant in proteins, fats, mineral elements and are a consolidated source of energy [7]. Being among the high ranked nut fruit crops, the world's annual production is 4.409 million tonnes out of an area around 1.305 million hactares [8]. Walnut plantations are generally derived from seedling origins (high heterozygosis and long gestation periods) which is undesirable [9]. Although it contributes to the large walnut gene pool, due to poor performance through vegetative propagation (grafting, budding and cutting) under natural conditions, multiplication of elite genotypes to a desired extent becomes unattainable [10].
Micropropagation has revolutionized the business of commercial nurseries in the last two decades. The Persian walnut is the most commonly cultivated and horticulturally established tree species for nut yield [11,12]. The potential of micropropagation of walnuts was initiated from explants, ranging from embryos to apical buds or petioles, having been extensively exploited a few decades back, since the first study on stem culture of black walnuts (J. nigra) [13]. Several authors have developed in vitro propagation protocols for various genotypes. However, recalcitrance of walnuts towards tissue culture have made it difficult for the micropropagation of newly developed genotypes [3,[14][15][16][17][18][19]. Moreover, genotype dependence, as well as the low reproducibility of these protocols, have made it's in vitro propagation more complicated. Most of these experiments focused on the browning phenomenon, elongation of shoots or buds, induction of roots, germination of embryos or somatic embryogenesis [6,20,21].
Rahman [22] studied the DKW medium and MS medium in which he found that DKW clearly showed dominating performance over MS medium in various tissue culture stages, such as callus mediated shooting and multiple shoot induction in Clitoriaternatea and Baptisia australis. Yegizbayevaet al. [23] also reported the best results on corrected DKW formulations for proliferation, rooting or ex vitro survival of walnut genotypes. Additionally, various techniques for inhibition of browning in microcuttings of English walnut have been examined, in which different media (DKW, DKW and MS) and antibrowning agents (activated charcoal and ascorbic acid) were employed for initiation of two cultivars, Chandler and Jamal. Activated charcoal supplemented medium of DKW was found to be the optimal medium, compared to the rest of the combinations [24].
Xiao-Dong et al. [25] focused on in vitro induction of the high-quality callus of Juglans regia L.cv, 'Xiangling', using different growth regulator levels in DKW basal medium. In their study, a DKW medium consisting of 1 mg/L BA, 2 mg/L KT, 250 mg/L glutamine, 500 mg/L casein hydrolysate, 200 mg/L Vc, 50 g/L sucrose and 2.8 g/L Gelrite was the best culture medium for induction of callus in immature cotyledons and embryos. Rout et al. [26] observed that browning of the medium resulted due to the exudation of polyphenolic compounds from the sliced end of rose explants, which could be suppressed by the addition of some important compounds, such as polyvinyl pyrolidone (PVP), citric acid, ascorbic acid, activated charcoal, and glutamine, or by frequently subculturing.
In vitro oxidative or phenolic browning of explants and media, in addition to other fungal and bacterial contamination, has become a major recalcitrant problem in the explant regeneration of walnut plants [27]. Different researchers have employed various antioxidant agents, viz., polyvinyl pyrolidone (PVP), activated charcoal and ascorbic acid, at varying concentrations to reduce the browning of media and tissues and have got varying results [28][29][30][31]. Keeping the above-mentioned constraints in mind, the aim of our research was to standardize the optimal in vitro medium and anti-oxidant regime for minimizing explant and medium browning.

Materials and Methods
The experiments were performed at the Commercial Tissue Culture Unit of Seven Star Fruits Pvt. Ltd., Aurangabad (MH) and the Tissue Culture Laboratory of Fruit Science Division, SKUAST-Kashmir, Shalimar, India, over the period 2019-2020. The investigation (Table 1) involved the study of different types of anti-browning treatments to explants and two media formulations to check in vitro browning of forced (treatments given to stock material to induce shooting under controlled laboratory conditions) and unforced shoot tips of "Sulaiman", a promising variety of walnut (J. regia L.). Actively growing unforced shoot tip explants 2-3 cm long were excised from mature walnut bearing trees of the Sulaiman variety, grown in the experimental walnutblock at the main campus of SKUAST-K, Shalimar, in the month of June-July 2019. Shoot tips were collected in flasks containing tap water, brought to the laboratory and washed thoroughly and kept under gushing tap water for 1 h. A.
Anti-browning agents For obtaining forced explants, pruned stock bud wood was collected from experimental field stock plants of the Sulaiman variety of walnut during the dormant months of November-December. In order to force ( Figure 1) the latent buds in the dormant wood (sub terminal or terminal potion, 15-20 cm long and 10-15 mm in diameter) were taken from bearing walnut trees. The dormant wood was first treated with Captan (@ 0.2 per cent), followed by cold storage at 4 ± 3 • C in poly-bags till further use. Forcing was done as per the procedure elucidated by [32].  The explant sterilization was done in 0.1% HgCl 2 for 5 min, which was followed by 70% ethanol treatment for 10s. After sterilization, the explants were washed 3 to 5 times under a laminar hood in sterilized water. Explants were inoculated on the medium by taking polarity into consideration and the buds were kept exposed. The culture tubes were then incubated at 25 ± 2 • C in a growth chamber with a 16/8 h photoperiod under light intensity of 3500 lux. Explants were shifted to fresh media after every 2-3 days of inoculation. Observations with regard to per cent explant survival (explants which showed signs of growth) and browned culture percentage were recorded within 3 ± 1 weeks of inoculation.
Two different types of the media i.e., MS [33] and DKW [34] media, were used in the present investigation. The composition of the different media utilized in the experiments, and details regarding stock solution preparations, are furnished in Table 2. The required quantities of sucrose, stock solutions of macronutrients, micronutrients, vitamins and anti-browning agents were dissolved in double distilled water as per treatment requirements [35]. The medium was adjusted to a pH of 5.8 with 0.1 N hydrochloric acid or 0.1 N sodium hydoxide, before adding agar to it. A gelling agent (Agar −7 g/L or Gelrite-2.8 g/L) was dissolved in boiled distilled water before adding it to the media and double distilled water was used to make the final volume. The hot media was poured into the test tubes and flasks just after boiling and, after tight plugging and wrapping with Al foil, the media were then autoclaved at 15 psi (121 • C for 15 min) [36].

Experimental Design and Statistical Data Analysis
The data generated from the experiments were subjected to statistical analysis (Analysis of variance) by using IBM-SPSS v.26.0,Chicago, IL, Statistical Software tool. The experimental model applied was of a completely randomized design. Treatments were replicated 3 times comprised of 24 explants per replication. Each container was the basic experimental unit; therefore, the average of all the containers was used as individual data for analysis.

Results
In the present studies in vitro browning was measured both on explants and nutrient media under the following headings:

Medium, Anti-Browning Regime and Explant Browning
The interaction effect of anti-browning agents, concentration and source of explant on percentage of browned explants was found to be significant. The minimum percentage of browned explants (13.6 ± 10.5 per cent) was recorded in forced shoot tips treated with ascorbic acid at 550 mg/L, while, the maximum explant browning (52.9 ± 5.2 per cent) was obtained in unforced shoot tips treated with Polyvinylpyrrolidone (PVP) at 150 mg/L ( Table 3). Table 3. Influence of anti-browning regime and explant source on explant browning (%) of walnut (Juglans regia L.) var. Sulaiman.

E1
E2  Table A1). F-Test (p ≤ 0.05) and R Squared = 0.932 (Adjusted R Squared = 0.898) were also performed. Table 4 shows how media and explant affected media browning percentage significantly. The highest percentage of media browning (44.6 per cent) was recorded in unforced shoot tips in MS medium, while the lowest value (26.4 per cent) was observed in forced shoot tips in DKW medium.Furthermore, the interaction effect between concentration and anti-browning agent on media browning percentage was found to be significant. Maximum media browning (58.4 ± 4.6 per cent) was recorded in PVP at 150 mg/L, while the minimum value (21.3 ± 1.3 per cent) was observed in ascorbic acid at 550 mg/L.   Table A2). F-Test (p ≤ 0.05) and R Squared = 0.978 (Adjusted R Squared = 0.967). Table 5 shows individual effects of explant and media on mean browning score per explant (0-4) at 5% level of significance. The maximum mean browning score per explant to the tune of 1.6 was recorded in MS medium, while the minimum value (1.4) was observed in DKW medium. The maximum mean browning score per explant to the tune of 1.7 was obtained in unforced shoot tips, whereas the minimum value (1.3) was observed in forced shoot tips. The interaction effect of concentration and anti-browning agent on mean browning score per explant (0-4) was found to be significant. The highest mean browning score per explant (3.4 ± 0.2) was recorded in PVP at 150 mg/L, while the lowest value (0.6 ± 0.1) was observed in ascorbic acid at 550 mg/L. Table 5. Influence of anti-browning regime, explant source and media on mean browning score per explant (0-4) * of walnut (Juglans regia L.) var. Sulaiman.

Medium, Anti-Browning Regime and Explant Survival Percentage
Theinfluence of four factorial interactions among anti-browning agent, concentration, source of explant and media on explant survival percentage was found to be statistically significant. The highest explant survival percentage to the tune of 74.5 ± 2.4 per cent was recorded in forced shoot tips cultured in DKW medium supplemented with ascorbic acid @ 550 mg/L, where as the lowest explant survival percentage to the tune of 25.8 ± 1.7 per cent was observed in unforced shoot tips in MSmediumsupplemented with PVP @ 150 mg/L (Table 6, Figure 2).   Table A4). F-Test (p ≤ 0.05) and R Squared = 0.982 (Adjusted R Squared = 0.973).

Discussion
In plant tissue culture, browning is the process in which explants release brown exudates or phenolic compounds into the medium from its cut surfaces in due course of dedifferentiation and/or re-differentiation [37,38]. Beruto et al. [39] reported the generation of phenolics by elevated activity of polyphenol oxidase (PPO). Moreover, phenylalanine ammonia lyase (PAL) enzyme converts phenylalanine to free phenolic substances for PPO [40]. Various authors have reported the involvement of PAL and PPO in the tissue browning of different plant species, such as sweet potato, cabbage, apple, pear, pineapple and herbaceous peony [41][42][43][44][45][46][47].
Different factors that affect browning are pretreatments, medium, subculture frequency, and temperature [48]. Charcoal adsorbs the phenols, thus causing the enzymes to be deprived of the substrates required for the generation of polymers which impart browning to the medium [49,50]. In addition, Fan et al. [51] reported the inhibition of PPO activity by NaCl, as well as by citric acid and ascorbic acid. Culture storage at low temperature was also found to enhance the activity of the PAL enzyme in walnuts [52].
A lower percentage of explant and media browning and higher survival percentage was found in the DKW medium as compared to the MS medium. The reason for this could be that the DKW medium contained a relatively lower quantity of nitrate compounds as compared to the MS medium (see Table 2), which could have induced the release of various yellowish brown phenolic exudates from the cut-off surfaces of explants [53]. These exudates, including juglone, in the medium interrupt cell growth, inhibit growth of the explant and also adversely affect their survival [26,54,55]. The superiority of the DKW medium over the MS medium could be attributed to its distinct composition of macronutrients, and also the Gelrite (gelling agent) in DKW is thought to be one of the key factors [22]. Compared to forced shoot tips, unforced shoot tips resulted in maximum percentage of explant and media browning and a lower survival percentage. This might be due to the high concentrations of phenolic compounds present in actively developing cells [56]. This variation in response may also be due to the relatively lesser inoculum load of the forced explants from dormant woods incubated in the growth chamber. However, the unforced explants from the unforced source grown in the open field conditions were exposed to the attack of different micro-organisms [57]. One more reason could be that during the process of forcing of explants in the growth chamber at a definite light and temperature regime, explants might have got acclimatized to in vitro conditions which have been otherwise found to influence polyphenol oxidase activity [58].
The higher percentage of explant survival and lower explant and media browning percentage was recorded in ascorbic acid at 550 mg/L, as compared to activated charcoal and polyvinylpyrrolidone at 50 mg/L. There could be two reasons for this. One reason is that when substrates lack PPO, ascorbic acid de-activates polyphenol oxidase (PPO) in an irreversible manner, probably through binding to its active site, preferentially in its oxy form. In the presence of PPO substrates ascorbic acid reduces PPO oxidized reaction products which results in a lag phase in PPO activity and reduces the dark product formation to a greater extent [59]. In addition, ascorbicacid at higher concentrations (350-1000 mg/L) performed as a prominent antioxidant and reduced the browning process to a greater extent [28][29][30]. However, the activated charcoal and PVP resulted in relatively poor results. They mainly act as adsorbents that bind phenolic compounds released from explant cultures and make them less toxic [60,61] but, at the same time, it has been observed that these compounds adsorb vitamins, growth hormones and nutrient ions, which might create nutritional and hormonal stress and result in lower percentage of survival and more browning in explants [60]. PVP has shown species specific effects in controlling the browning process [62], which may be due to the presence of a wide variety of phenolics in different plant species. The significance of anti-browning agent, concentration, explant and media on explant survival percentage might be due to the positive synergistic effects of these factors on the growth of the explants.

Conclusions
Based on emanated results of the present experiments it may be concluded that forced shoot tips along with DKW medium were found to be the best in terms of least browning and highest survival percentage, as compared to unforced shoot tips grown in MS medium. Anti-browning agent, ascorbic acid at 550 mg/L, showed the greatest response in terms of least browning and the highest survival percentage of explants.