Introduction

The Morus sp (mulberries) are flowering and deciduous plants of Moraceae family. Mulberries have been domesticated over thousands of years and are cultivated in Asian, European, American, and African continents (^{Özgen et al., 2009}). There are many applications to mulberries, which include livestock feeding (^{Sánchez, 2000}; ^{Milera et al., 2010}; ^{Islam et al., 2014}) gardening or landscaping (^{Ljubojević et al., 2023}), environmental safety (^{Mallick & Sengupta, 2022}), and natural products (^{Özgen et al., 2009}; ^{Mallick & Sengupta, 2022}). Their multiple applications led to great exploitation worldwide, from smallholders to large-scale production (^{Sánchez, 2002}). The impressive adaptability of mulberries comes from their simple cultivation, fast growth, and survival under challenging environments (^{Rahman & Islam, 2021}).

The main agronomic application of mulberries is for leaf harvest and thus feeding the silkworm (Bombyx mori) under sericulture or silk farming (^{Özgen et al., 2009}; ^{Bobis et al., 2018}; ^{Mallick & Sengupta, 2022}). Importantly, mulberries leaves are the sole food provided to silkworms (^{Kumar et al., 2012}; ^{Rahman & Islam, 2021}). Data from multiple reports indicated that fruits and leaves have high content of crude protein, sugars, fatty acids, micronutrients, and dry-matter digestibility, which supports its multiple applications (^{Bamikole et al., 2005}; ^{Liang et al., 2012}; ^{Koyunco et al., 2014}; ^{Zach et al., 2017}). However, mulberries require adequate management practices to provide such high nutritional content (^{Benavides et al., 1994}). Mulberry are deciduous trees and their leaf production is destined to feeding the silkworm larvae, however through winter and under plague and disease attack thous yield is diminished and the leaves quality reduce (^{Banday et al., 2017}).

In vitro culture of plant tissues allows large-scale propagation of desirable genotypes under sterile conditions (^{Sharma & Thorpe, 1990}; ^{Sil, 2021}). Such approaches usually rely on young explants for in vitro propagation, since there is greater viability (^{Acuña, 2020}), alongside the use of plant growth regulators (^{Freire-Seijo, 2003}; ^{Ikeuchi et al., 2013}; ^{Geetha & Murugan, 2017}). In vitro mulberry calli culture is strongly influence by the addition of plant growth regulators to the culture media. The auxin and cytokinine stimulate necessary reactions for the calli, steam or rooths in vitro culture from an explant (^{Ikeuchi et al., 2013}). ^{Bhau y Wakhlu (2001)} report the use of 2,4-dichlorophenoxyacetic acid (2,4-D) and benzyladenine (BA) (1.0 mg L^-1 y 0.5 mg L^-1) successfully in the propagation of mulberry calli from leaf explants, internodes and stalk.

The in vitro propagation of mulberries calli offers the possibility of feeding silkworm larvae continuously. The utilization of sterile mulberry calli production as a model in silkworm larvae feeding guaranty the availability of silkworms as a research organism model in further applications (^{Ashraf & Qamar, 2023}).

The aim of this study was to determine the effect of variety alongside plant growth regulator supplementation on in vitro propagation of white mulberries (Morus alba L.) calli and its application as a supplementary feed for growing silkworm larvae.

Material and methods

Location and vegetal material

The project was developed according to institutional and national legislation. The experiments were performed in the plant tissue culture laboratory at the Departamento de Fitotecnia, Universidad Autónoma Chapingo, located in Texcoco, Mexico in 2014.

The vegetative material was obtained from two years old white mulberry trees (Morus alba L.) of the SLP3 and SLP5 varieties grew and acclimated in a greenhouse. The white mulberry characteristics were SLP3 larger ≈20cm softer (pliable) and thinner leaves and SLP5 smaller ≈15cm visibly thicker and brittle leaves.

Plants were placed in 40 x 40 cm polyethylene pots filled with 3 to 6 mm tezontle substrate and were irrigated with 250 ml tap water twice a day and supplemented weekly with a 3 ml L^-1 Byfolan^®, 0.5g L^-1 Aliette^® and 1.5g L^-1 AgrimyCu^® solution by sprinkled to control contamination in plants. Plants were cultivated by 1.5 years.

In vitro growth leaves culture

The in vitro culture was established with 20 SLP3 and 20 SLP5 young leaves (seven to eight cm length) that were washed with Roma^® detergent (5g L^-1) and Tween^™-80 (Fisher Chemical) hi-purity (1 ml L^-1) in distilled water for 20 min. Further, leaves were surface disinfest with 70% ethanol for three minutes, and subsequently in a solution of 10 ml•100 ml sodium hypochlorite (Cloralex^®). Leaves of mulberries were transferred to Petri dishes and rinsed three times with sterile ultra-purify water.

The culture medium to in vitro propagation was based on a modified version of the MS medium (Sigma Aldrich ^®) (^{Murashige & Skoog, 1962}) supplemented with 7.0 g L^-1 Meyer ^® agar (≥99%), 3% sucrose (99.5%) (Sigma Aldrich ^®), 0.4 mg L^-1 thiamine (≥99%) (Sigma Aldrich ^®), and 100 mg L^-1 myo-inositol (≥99%) (Sigma Aldrich ^®). The pH of the solution was adjusted to 5.7 + 0.1. 20 ml the culture medium was placed in (200 ml) Gerber^® flasks, which were sealed with polyethylene tops, and sterilized by autoclaving at 121°C, 15 PSI for 40 min. Afterwords the flasks were set at room temperature 22 ± 1°C and the plant growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) (≥95%) and thidiazuron (TDZ) both from Sigma Aldrich^® were added as is described in experimental design treatments.

Leaves rinsed in sterile ultra-purify water were dissected with scalpel in three portions: apical, medium and basal acording to ^{Freschi et al. (2010)}. Each leaf portion were placed randomly in Gerber^® flasks with culture medium MS for each treatment. Flasks were sealed to avoid contamination and placed in a dark growth chamber at 22±1°C, culture was performed for 60 days.

Explants decontamination protocol

The explants exposed to contamination were retrieved from in vitro culture and submerged in a Roma^® detergent (5g L^-1) and Tween^™-80 (Fisher Chemical) hi-purity (1ml L^-1) solution in distilled water. Subsequently, explants were submerged in 70% ethanol for three min, submerged in 10 ml•100 ml sodium hypochlorite (Cloralex^®) solution for 15 min, and placed with 4% calcium hypochlorite for 15 min. Additionally, explants were exposed to a 100% Timsen^® solution for 15 min, Benlate^® and Fungimycin 100^® 2g L^-1 solutions for 10 min, and rinsed in 100% Protect^® H₂O₂ before being placed in a Petri dish containing MS medium.

Experimental Design

To determinate the effect of mulberries (Morus Alba L.) variety and supplementation of plant growth regulators on in vitro mulberry calli propagation, a completely randomized design was performed with four treatments: i) 1 mg L^-1 2,4-D/ 3 mg L^-1 TDZ, ii) 3 mg L^-1 2,4-D/1 mg L^-1 TDZ, iii) 1 mg L^-1 2,4-D/1 mg L^-1 TDZ and vi) 3 mg L^-1 2,4-D/3 mg L^-1 TDZ with five replicates; each flask was an experimental unit. There were evaluate weight and volume as non-destructive measurements (quantitative variables) and visual coloring as qualitative variable for in vitro grown calli, during the culture period in 14 days intervals.

Evaluation of in vitro grown calli to feed Bombyx mori larvae

The unhatching silkworm eggs were obtained from the Universidad Politécnica Francisco I. Madero, Tepatepec - Hidalgo, Mexico and were maintained at room temperature at 22±1°C in the plant tissue culture laboratory until larvae hatching.

Five silkworm larvae were placed in 10 cm disposable Petri dishes at room temperature at 22 ±1°C immediately after hatching. The explant in vitro grown and calli were used to feed the silkworm larvae totally the first 21 days, twice a day (9:00 and 19:00 h). The effect of feeding with in vitro grown calli of white mulberries was determined by measuring the weight and length of silkworm larvae at seven days intervals. The silkworm larvae survival percent was determined at the end of the 21 days experiment. Subsequently the larvae were feed 25 days more with mulberry leaves until the cocoon weave at day 46 and the emerging of the moth after 12 to 15 days after.

Statistical analysis

The variables weight and volume of the in vitro grow mulberry calli and the length and weight of the silkworm larvae feed with in vitro grow calli were analyzed with an analysis of variance (ANOVA) and the post-hoc analysis was done with Tukey's mean test at 5% significance level with the software Statistical Analysis System ^® (SAS) 9.0.

Results and discussion

In vitro propagation of white mulberries calli from leaves explants

The differences in development of white mulberries calli in vitro became evident from week two onward (Figure 1).

The treatment T4 and T2 displayed the best traits, which included calli weight and volume (Table 1) (p≤0.05). Under the comparison between varieties of white mulberries, the variety SLP5 was different in weight and volume than the variety SLP3, (p≤0.05) (Table 2).

Figure 1 In vitro culture of Morus alba L. calli from leaf explants. a-c) Second, fourth, and sixth week after in vitro culture respectively; d-e) Eighth week; f) Enlarged view of calli during the eighth week of culture. Scale barr 0.5 cm. 

However, the portions of leaves did not affect significantly in vitro growth (Table 3). The in vitro grown calli displayed friable texture, separately from the treatment. Further, most in vitro grown calli of white mulberries were spongy-like and granular. The color of in vitro grown calli changed during their growth (Table 4), gradually mulberry calli acquired a darker coloring due the natural tissue oxidation (Figure 2).

Figure 2 Color of Morus alba L. calli under in vitro conditions. a) Fourth week of culture. b) Seventh week of culture. c) Necrosis in calli at the eighth week of culture. Scale barr 1 cm. 

Plant growth regulators supplementation with 2,4-D was the best approach for growing in vitro calli of white mulberries. The culture conditions are paramount for plant regeneration during in vitro propagation (^{Taha et al., 2020}), while much progress was made in this technology for mulberries (^{Thomas, 2002}; ^{Vijayan et al., 2011}; ^{Taha et al., 2020}). Plant growth regulators are instrumental to calli induction and plant regeneration under in vitro conditions (^{Freire-Seijo, 2003}; ^{Geetha & Murugan, 2017}). ^{Ikeuchi et al. (2013)} mention that the auxin and cytokinine stimulate necessary reactions for the calli, steam and roots in vitro cultured from an explant. Several growth regulators (e.g., auxins, cytokinins) have been tested for in vitro propagation of mulberries (^{Geetha & Murugan, 2017}; ^{Fonseca-Carrasco et al., 2020}; ^{Taha et al., 2020}). The most prominent role of 2,4-D was similar to several previous studies (^{Pierik, 1990}; ^{Bhau & Wakhlu, 2001}; ^{Cholo & Delgado, 2011}; ^{Espinosa et al., 2012}), thus demonstrating its prominent role of the growth and development of mulberries.

During experiment the grow in vitro explants get contaminated with fungi and bacteria from 120 flasks get contamined 13 flasks (11%) and was recuperated 10 flasks (80%) by the decontamination protocol. In ^{Hernández & González (2010)}, mentioned that the season of the year and the collection procedure are a frequently origin of contamination and made emphasis in the need of developing decontamination protocols for in vitro culture plant tissues.

Feeding silkworm larvae with white mulberries calli

The feeding silkworm larvae with in vitro grown white mulberry calli with different concentration of plant growth regulator affect the larvae growth, denote a bigger weight and length in T4 (Table 5).

The silkworm larvae stage has a variable duration that depends from the environmental conditions; at 20 °C the cycle length is about 50 to 55 days (^{Rodríguez et al., 2012}), in this research the larval stage showed a 45 days duration in normal conditions at room temperature at 22±1°C (Figure 3). The survival of silkworm larvae was 57.14%, from this, 23 larvae, 100% weaved silk cocoons emerge into moths. Silk cocoons had a mean length of 2.54 ±0.20 cm and a mean diameter of 1.315 ± 0.09 cm (Figure 4).

Figure 3 Feeding silkworm (Bombyx mori) larvae with in vitro grown calli of Morus alba L. a -b) First larval stage. c-d) Fourth larval stage.Figura 3. Alimentación de larvas de gusano de seda (Bombyx mori) con callos cultivados in vitro de Morus alba L. a-b) Primer estadio larvario. c-d) Cuarto estadio larvario. 

Figure 4 Cocoons obtained from adult silkworms (Bombyx mori) fed for 35 days with in vitro grown calli of Morus alba L. varieties SLP5 (a) and SLP3 (b). Scale barr 1 cm.Figura 4. Capullos obtenidos de gusanos de seda adultos (Bombyx mori) alimentados durante 35 días con callos cultivados in vitro de Morus alba L., variedades SLP5 (a) y SLP3 (b). Barra de escala de 1 cm. 

The fact that silkworm feeding relies solely on mulberries makes it susceptible to undersupply if the plant faces environmental challenges or incidence of disease and blight (^{Kumar et al., 2012}; ^{Caccam & Mendoza, 2015}; ^{Rahman & Islam, 2021}). Therefore, technology alternatives that safeguard the feeding supply for silkworms is highly desirable. As far as is known, the in vitro grown mulberries have not been tested for feeding silkworms. Under such scenario, in this research the in vitro propagation of mulberries calli were feeding acceptable by silkworm larvae under sterile conditions (^{Sharma & Thorpe, 1990}; ^{Sil, 2021}).

The white mulberry SLP5 variety was the most productive between those tested here for in vitro growth of calli. There is vast evidence that mulberry variety plays an important role on in vitro propagation efficiency (^{Bhatnagar et al., 2001}; ^{Taha et al., 2020}). These results were similar to previous reports on the growth rate of plants (^{Rodríguez-Ortega et al., 2013}), thus suggesting that in vitro growth may mirror the in vivo growth kinetics among varieties.

Conclusion

In this research the in vitro propagation of white mulberry was independent on plant variety. White mulberry in vitro growth calli under treatment four and two (3mgL^-1 2,4-D/3mgL^-1 TDZ and 3mgL^-1 2,4-D/1mgL^-1 TDZ) performed the best characteristics in the evaluated variables. The in vitro propagation of mulberries calli were acceptable as food by silkworm larvae under laboratory conditions.