The Effect of Slow-grown in Vitro Storage Under Different Light Spectra on Banana Plantlets Cv. Prata Catarina (AAB).

16 Maintaining updated in vitro plant subcultures is essential for commercial micropropagation and tissue culture research. In unusual situations, the subcultures can be delay and the slow-growth in vitro storage technic could be applied to reduce the loss of plant material. The present study aimed to evaluate the slow-growth in vitro storage 19 of banana plantlets (‘Prata Catarina’; group AAB) under different light spectra. Shoot cultures in MS medium without 20 plant growth regulators were maintained under blue (B), red (R), red plus blue (R2B), and white (CW) light spectra 21 (25°C ± 2°C; 50 µmol m -2 s -1 ) for up to 140 days. The plantlets maintained under the R, CW, and R2B spectra did not 22 survive after 140 days of in vitro slow-growth storage. The plantlets maintained under the B spectrum survived after 23 140 days of in vitro


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In commercial micropropagation laboratories (CML), operational difficulties may arise during commercial 30 micropropagation processes when the in vitro-produced plantlets that are ready to be transplanted cannot be moved to 31 the greenhouse because they are full of unsold plants or because the customer is not ready to receive the plants. Such 32 operational difficulties can result in financial losses to both CMLs and customers (Watt et al. 2000). Recently, many 33 CMLs and tissue culture research centers (TCRC) were temporarily closed due to the COVID-19 pandemic, which 34 reduced the workforce, thereby limiting the subculture frequency during the lockdown. In such unusual situations,

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CMLs and TCRCs can employ in vitro slow-growth storage (SGS).

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During in vitro SGS, to increase the period before subcultures, the development of plants is retarded by 37 suppressing their metabolism, albeit without altering the genetic uniformity and quality of micropropagated plantlets 38 (Kamińska et al. 2016). Culture conditions (temperature, photoperiod, and light intensity) and culture medium 39 components (organic and inorganic nutrients, osmotic, and plant growth regulators) can be adjusted to suppress plant    cytokinin stops working, and the shoots start rooting until consuming all the culture media. In this case, the laborers 50 spend triple times working to cut the roots, and the frequency of contamination increase (Pers. Comm).

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To date, few studies have explored different light spectra for in vitro SGS. The use of different light spectra 52 for in vitro SGS of Heliconia champneiana 'Splash' plantlets was evaluated and promising outcomes were achieved       were compared with Tukey's test at 5% significance.

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During SGS, there was an interaction between plant height and light spectrum (p = 0.0359) (Fig. 1a). The 91 cigar leaf length of explants increased steadily up to I4 but decreased during I5 (Fig. 2c). The height of explants grown 92 under different light spectra was in the order B<R2B<CW<R (from the shortest to tallest) across time intervals (Fig.   93 1a).

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During SGS, the different light spectra affected leaf number (p = 0.0005) and pseudostem thickness (p = 95 0.0285). These characteristics steadily increased until I5 (Fig. 1b and 1c). No symptoms of leaf senescence were 96 observed under any light spectrum at I5 (Fig. 2a). The different light spectra affected fresh weight (p < 0.0001). During 97 the experimental period, fresh weight was the lowest under the B spectrum, and the value fluctuated under the R 98 spectrum during I4. In contrast, fresh weight was the highest under the CW and R2B spectra from I2 to I5 (Fig. 1d).

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During SGS, there was an interaction between root number and light spectrum (p < 0.0001). During I2, there 100 was a significant increase in growth under the R2B spectrum compared with that under the remaining spectra (Fig.   101 1d). The number of primary roots increased from I2 to I4 under all evaluated spectra, except R2B.

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There were significant differences in evaluated root number under the CW and other spectra during I3 and 103 I4. During I5, primary root number under the CW spectrum and growth under the R2B and R spectra were significantly 104 reduced, but the values were stable under the B spectrum. Of note, under the CW spectrum, primary root number 105 increased from I2 to I4 but decreased during I5.

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There was a significant interaction between the length of the longest root (Fig. 1e) and the light spectrum (p 107 < 0.0001). This length increased from I1 to I4 under all spectra, except CW. Under the CW spectrum, there was a 108 significant reduction in this length from I2 to I3 and a further significant reduction from I3 to I4, and the values were 109 lower than those under all the remaining spectra. During I5, there were significant reductions in the length of the 110 longest root under the R, B, and R2B spectra, but the values were stable under the CW spectrum. The roots suffered 3 the effect of in vitro senescence over time, in the order of CW>R2B>R>B. Under the B spectrum, characteristic young 112 root color was observed even after 140 days of in vitro SGS (Fig. 2a and red rectangle in 2b). Although we did not 113 quantify the consumption of the culture medium, a larger volume of the culture medium, which was 114 colorless/translucent even during I5, was remaining under the B spectrum.

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All plants survived SGS until I4 (Fig. 1g). The explants were grown under the CW, R2B, and R spectra died 116 soon after removal from the test tubes. Excessive browning resulted in the death of some explants. At the intersection 117 of the pseudostem and root, tissue breakage occurred during explant wash, subsequently leading to death, under the R

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There was a significant interaction between the SPAD index and the light spectrum (p < 0.0001). The trends 120 were similar under the CW, R2B, and B spectra, with a lower index recorded under B; the opposite trends were 121 observed during I1 and I2, but they remained stable from I3 to I5 (Fig. 1h).

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The present study demonstrated that different light spectra affected all evaluated parameters during in vitro 128 SGS. Regarding plantlet height, there was a reduction from I4 to I5 under all spectra, which was caused by the 129 shortening of the cigar leaf. In a previous study, the effects of the B and R spectra and their different combinations 130 during in vitro cultivation Musa paradisiac 'Nam Dinh' were assessed using a sugar-free technique with CO2 injection 131 (Nhut et al. 2002). In that study, the plant height was in the order of B < PGF (fluorescent lamp) < R2B (70% red +

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The effect of light quality on the cellular redox balance in vitro has been poorly studied and is restricted to

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The lowest SPAD index was observed from I2 to I5 under the B spectrum, indicating lower photosynthetic 175 activity and, consequently, lower metabolic activity during SGS. These results are similar to those reported previously 176 for 'Nam Dinh', in which blue light completely inhibited plantlet growth (Nhut et al. 2002).

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The acclimatized plants showed no morphological variation. In the present study, plants under the B spectrum 178 showed reduced growth during I5 of the acclimatization period. In a previous study, no differences in the height, fresh