Next Article in Journal
Calcium Involved in the Enrichment of γ-Aminobutyric Acid (GABA) in Broccoli Sprouts under Fructose Treatment
Next Article in Special Issue
A Simple but Effective Combination of pH Indicators for Plant Tissue Culture
Previous Article in Journal
Atmospheric Vapor Impact on Desert Vegetation and Desert Ecohydrological System
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 12
Issue 2
10.3390/plants12020225
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm

by
Ravish Choudhary
1,*,
Surendra Kumar Malik
2,*,
Rekha Chaudhury
2 and
Atmakuri Ananda Rao
3
1
Seed Science and Technology, ICAR-Indian Agricultural Research Institute, Pusa Campus, New Delhi 110012, India
2
Tissue Culture and Cryopreservation Unit, ICAR-National Bureau of Plant Genetic Resources, Pusa Campus, New Delhi 110012, India
3
Central Sericultural Germplasm Resources Center, Hosur 635109, India
*
Authors to whom correspondence should be addressed.
Plants 2023, 12(2), 225; https://doi.org/10.3390/plants12020225
Submission received: 18 November 2022 / Revised: 23 December 2022 / Accepted: 30 December 2022 / Published: 4 January 2023
(This article belongs to the Special Issue Advances and Applications in Plant Tissue Culture)
Browse Figures
Review Reports Versions Notes

Abstract

:
A two-step freezing cryoprotocol preceded by desiccation to 15 to 25% moisture content was developed and successfully applied to winter dormant buds of mulberry (different Morus spp.) of a core set comprising 238 accessions studies in our laboratory. The survival and recovery percentage of diverse accessions cryobanked for various periods were tested under in vitro conditions, and several factors were analyzed to determine their role in optimizing the recovery of low-viability accessions. The effect of rates of freezing and thawing (both fast and slow), were tested and recovery compared. Recovery conditions such as dark incubation and rehydration in sterile moist moss grass for different durations after cryopreservation led to a higher survival percentage compared to controls. Two different recovery culture media were compared for their efficiency in survival. On average, the survival under in vitro culture conditions using optimized conditions was high: above 60% in majority of the accessions. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. The recovery percentage of winter dormant buds after cryopreservation via slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range from 63.3 to 90.9% with an average of 81.05%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. Regeneration of cryopreserved mulberry germplasm after 6 years of storage indicated no survival loss over different years of storage, and 33–40% of the accessions showed viability above 40%, up to a maximum of 100%. Maximum shoot formation (100%) was obtained from Morus alba. The majority of the accessions were rooted in vitro within 20–25 days of subculture in the auxin rich rooting media, except in wild species M. latifolia and M. laevigata, which took longer (45 to 60 days) for root development. All the rooted plantlets were then transferred to the field and successfully established in a glasshouse.

1. Introduction

Mulberries are one of the healthiest foods among horticulture crops, and there has recently been an increase in interest in less well-known horticultural plant species such as these. Mulberry fruits are scrumptious, healthy, and offer a host of remarkable health advantages. Less-known fruits are accepted as insurance in the future horticultural cultivation in the climate-change scenario due to their resistance to abiotic and biotic conditions [1,2,3]. Mulberry (Morus L., Family Moraceae) that originated at the foothills of the Himalayas is an economically important woody tree species which is extensively used for rearing silkworms. It is an out-breeding, heterozygous, and perennial tree species also used in agro-forestry and horticulture [4,5,6]. Sanjappa [7] recognized 68 species within the genus Morus and in India, 4 main species of Morus, viz., M. indica, M. alba, M. laevigata, and M. serrata have been reported [8]. The Central Sericultural Germplasm Resources Center (CSGRC), Hosur, Tamil Nadu, (India) is the National Active Germplasm site for mulberry germplasm in India, which conserves 13 Morus species from 26 different countries, totaling 1120 accessions in their field gene-bank [9]. The cryopreservation of dormant buds of mulberry germplasm as an economical, safe, and effective method of long-term conservation has been reported as a safety back up of field gene-banks. The cryopreservation has proved its worth for ensuring for long-term conservation and utilization of plant genetic resources [10,11,12,13,14]. The majority of wild Morus species showed sensitivity in nature and demonstrated species-specific variation under in vivo and in vitro conditions [14]. Some wild mulberry germplasms such as M. tiliaefolia and M. serrata showed significantly less recovery after cryopreservation [14,15]. However, success can be judged only when high recovery rates are achieved after cryostorage. At the National Bureau of Plant Genetic Resources (NBPGR), New Delhi, 238 accessions of the core set of mulberry germplasm are maintained at National Cryogenebank in the form of dormant buds from −170 °C to −180 °C temperature [15]. These buds need to be recovered at the times of need for raising complete plantlets. In the present study, the cryobanked winter dormant buds of mulberries were recovered using different variables to optimize the recovery conditions via an in vitro culture method for subsequent field transfer.

2. Results

The effect of various variables, as tested by each individually or in combination, was recorded in terms of recovery percentages.

2.1. Effect of Harvest Dates

Buds of more than 50 accessions harvested during the months of December, January, and February for three consecutive years were checked for initial moisture contents. Moisture content was found to range from 24.2 to 61%. Accessions were categorized into each of the moisture content (MC) ranges and expressed as percentage values (Figure 1). It was observed that a large proportion (91%) of December harvested buds had MC range values of 36–55% and 9% showed high moisture content (above 55%). January harvested buds also showed similar results, but 5% of the samples showed a slightly lower value of moisture content (26–35%). More than 95% of February-harvested buds showed moisture content in the range from 26 to 55%. Temperatures during December, January, and February months ranged from a maximum of 25–27 °C to a minimum of 14–15 °C and nil or negligible rainfall. Based on these observations in subsequent experiments, buds were harvested only during mid-February for cryopreservation.

2.2. Relation between Desiccation Level and Survival of Buds after Cryopreservation

Moisture content of freshly harvested dormant buds of different Morus species ranged from 28.1 to 59.6% with an average of 47.5%, and initial viability was found in the range from 46.7 to 100% with an average of 75.1%. Moisture content after 4–5 h desiccation in charged silica gel ranged from 9.5 to 17.8%, with an average of 15.2% observed in the present study. Winter dormant buds after removal of 5–8 outer scales were tested for viability before and after cryostorage. The viability of dormant buds before cryopreservation after desiccation ranged from 25 to 100% with an average of 51.5%. After cryostorage, no significant change in the viability was noticed. Dormant buds showed viability in the range of 25 to 100% with an average of 50.4%. Maximum viability (100%) was found after cryopreservation in the accessions of Morus indica × M. alba (IC 313836) at 17.3% of moisture content after 5 h desiccation followed by M. indica (IC 313703) (95%) at 15.31% moisture content and M. alba (EC 493822) (80%) at 16.11% moisture content. Minimum viability (25%) was recorded in M. rubra (EC 493988) at 15.5% moisture content, followed by M. latifolia (EC 493831) with viability (28.6%) at 9.5% moisture content and M. cathayana (EC 493775) with viability (30%) at 13.8% of moisture content (Table 1). The seedlings obtained from cryostored dormant buds were visually normal and healthy. A total set of core collection of 13 diverse species of mulberry germplasm from different parts of India have been successfully cryostored as a base collection in the cryogenebank (Figure 2).

2.3. Effect of Slow, Fast, and Two-Step Cooling Rate

The recovery percentage of winter dormant buds after cryopreservation through slow freezing and slow thawing with rehydration by moist moss grass for 2 h was recorded in the range of 63.3 to 90.9% with an average of 81.1%. Without rehydration, it ranged from 50 to 75% with an average of 60.4%. The recovery was found through slow freezing and fast thawing with rehydration in the range of 7.7 to 52.4% with an average of 35.8%. Without rehydration, this ranged from 0 to 33.3% with an average of 18.7%. However, the recovery of dormant buds after cryostorage through fast freezing and slow thawing with rehydration was obtained in the range from 11.1 to 36.4, with an average of 23.8%. Without rehydration, this ranged from 3.3 to 16.7% with an average of 10.7%, while recovery showed fast freezing by fast thawing with rehydration in the range of 6.7 to 25.4% with an average of 16.3%. Without rehydration, this ranged from 0 to 16.7% with an average of 7.5%. The highest recovery (90.9%) was found in the accession of M. indica (IC 313887) after cryopreservation by slow freezing and slow thawing with rehydration in moist moss grass for 2 h at room temperature. Without rehydration, recovery was found 75% in M. alba (IC 313736). In the case of slow freezing followed by fast thawing, the higher recovery was found 52.4% with rehydration and 33.3% recovery was observed without rehydration in M. indica (IC 313977). However, in case of fast freezing followed by slow thawing, the maximum recovery was recorded 36.4% with rehydration in M. indica × M. alba (IC 493875), while without rehydration, it was found 16.7% in M. indica. Meanwhile, 25.3% recovery was found after fast freezing followed by fast thawing and rehydration, and without rehydration, the recovery was 16.7% in M. indica × M. alba (IC 493875) (Table 2).

2.4. Effect of Thawing and Rehydration

The viability percentage of dormant buds was found to be at its maximum between moisture contents of 15–25%. Above 25% and below 15% of moisture content, the viability of buds gradually decreased in cases with slow freezing and fast freezing. In the case of fast freezing using fast thawing, the viability of buds above 50% of moisture content was lost. However, during slow freezing, a maximum 80% viability was observed, while in case of fast freezing, 70% was found at 19% moisture content (Figure 3).

2.5. Effect of Recovery Media

The percentage of shoot formation ranged from 26.7 to 100% with an average of 71.7% in TDZ (0.1 mgL−1) while in case of BAP (1 mgL−1), it was between 33.3 and 100% (Table 3). The viability was a maximum of 100% with the help of BAP in M. indica (IC 314255) and the minimum was 33.3% in M. alba (IC 314257) while for TDZ, maximum viability (100%) was obtained in M. indica (IC 314255) and the minimum was 26.7% M. alba (IC 314257). With the exception of two accessions, IC 405829 (M. indica) and IC 405800 (M. bombycis), BAP (1 mgL−1) generally outperformed TDZ (0.1 mgL−1) in terms of viability percentage, with IC 405829 (M. indica) and IC 405800 (M. bombycis) showing much higher viability than BAP (1 mgL−1). After retrieval, the buds started sprouting in vitro after 3–4 week of culturing (Figure 4A). Once the bud sprouting started, the shoot formation, rooting, and hardening (Figure 4E–H) was normal. After elongation, complete plantlets were transferred for hardening in the field condition. The initial growth of frozen meristems was extremely slow compared to unfrozen buds. However, leaves began to expand within 2 weeks and shoot development took place during 3–4 weeks in culture. In the current study, the shoots derived from cryopreserved winter buds produced roots in MS medium supplemented with 0.1 mgL−1 NAA and, after hardening in pots filled with vermiculite and peat moss (2:1) in a humified atmosphere, they were transferred to soil.

2.6. Effect of Dark Incubation

Buds that did not show any recovery following direct culturing under light condition showed viability of 12–100% with an average of 52% in all the different Morus species when culture conditions were modified by dark incubation. The highest recovery (100%) was recorded after modified conditions in M. latifoila (EC 493779), while minimum was 12% in M. alba (EC 493799) (Table 4).

2.7. Viability after Cryostorage

The viability percentage was initially found to be 57.7% (the average of eight accessions of different Morus species) after 3 months of cryostorage. However, no significant loss was observed in terms of viability percentage after 6 years of cryostorage, which was found to be 54.2% (Table 5).

3. Discussion

Dormant buds are subjected to cold acclimation. Plants exposed to naturally cold, non-freezing temperatures have high tolerance to subsequent artificial freezing, enabling them to be cryoconserved, ensuring long-term viability. Several physiological and biochemical mechanisms are known to confer this natural tolerance. Difference in hardiness to low temperatures in nature is observed between shoot tissues, with bud and cortex being the most hardy tissue and xylem being the least hardy [16]. Tolerance of such acclimated dormant buds to a much lower temperature of liquid nitrogen, however, depends on extracellular freezing, which is caused by slow stepwise freezing. This was studied in mulberry [14,17,18,19], in almond [20], in apple [21], in pear, in elm [13], etc. The techniques, however, so devised ultimately depend on successful higher recovery.
Most species are reliably preserved after being desiccated to 20-30% [22] and then cooled slowly between −30 and −40 °C before exposure to the vapor phase of liquid nitrogen [16,23,24]. In the present study, Morus indica, M. alba, M. bombycis and M. sinensis showed high growth values when pre-desiccated to moisture contents between 15 and 25%. After two-step freezing, high viability was observed. It was also observed that most of the accessions, which survived LN exposure, had inherent dormant or semi-dormant nature, whereas accessions that failed to recover after LN exposure were non-dormant or semi-dormant in nature (Table 1). It seems there is a correlation between the dormancy of the plant and freezing tolerance leading to recovery after LN exposure. The values of moisture content and in vitro survival fresh, after desiccation and cryoexposure were shown in Table 1.
Tyler and Stushnoff [21] mentioned that the desiccation rates are significantly correlated to the size and compactness of the buds. Niino et al. [25] found that in winter mulberry buds, with small vascular tissues, partial dehydration prior to pre-freezing improved shoot formation in meristem culture of cryostored buds after thawing. In Malus species, high survival can be obtained with nodal segments, whereas the moisture content is reduced from 45% (moisture content when collected) to 25 to 30% prior to cooling [26]. Pear buds had the best survival at 41% [27]. In our study, dormant buds of Morus species showed higher survival when the moisture content was reduced by 15 to 25%. As well as increasing the desiccation, the buds decreased the moisture content and viability before and after cryopreservation.
The last pre-freezing temperature (just before plunge in liquid nitrogen) required for survival after cryostorage depends on the thawing method. Suzuki et al. [27] found that higher rates of shoot formation were obtained at −30 °C pre-freezing temperature, followed by slow thawing in the pear germplasm, and similarly in apple [21,28]. However, in mulberry, higher survival was obtained at −20 °C, followed by rapid thawing in a ±37 °C water bath (3–5 min) [29]. The simplest and quickest pretreatment for the successful cryopreservation of mulberry buds was a 24 h exposure of the samples to each lowering temperature (−5 °C/day) before being plunged in liquid nitrogen and subsequent rapid thawing ±38 °C [29]. During freezing, water migrates from the super cooled shoot primordial tissue to the bud scales and the dehydrated shoot tips attain a high degree of freezing tolerance, preventing intracellular freezing and lethal ice nucleation [10]. Pre-freezing rate in combination with the last freezing temperature as well as thawing methods was equally factors in improving the shoot formation rates of cryopreserved buds. Pre-freezing with a daily decrease of −5 °C in temperature gave good results compared to decreases of −10 °C [30]. It is important to dehydrate the tissues to prevent lethal intracellular cooling prior to rapid freezing to produce higher survival after cryopreservation [18]. Yakuwa and Oka [29] also reported that meristems excised from segments without pre-freezing and transferred directly from 0 to −196 °C never survived.
In our study, attempts were made to compare the role of thawing in recovery rates of buds cryopreserved by two rates of cooling, namely, slow and fast. Data obtained for accessions of different Morus species are shown in Table 2. Buds conserved by slow freezing followed by slow thawing with rehydration showed a high recovery percentage (avg. 81.1%), while 60.4% recovery was recorded without rehydration. Using fast thawing for buds conserved by slow freezing led to a lower percent recovery of 35.8% and 18.7%, respectively. In contrast, using fast freezing, slow thawing led to recovery of 23.8% and 10.7% for rehydration and non-rehydrated buds, respectively. However, using fast freezing and fast thawing with rehydration recovery, the percentage was very low (avg. 16.3%). Such buds without rehydration had much lower percentages at 7.5% (Table 2).
Touchell and Walters [31] found that embryos of Zizania palustris were rehydrated for 60 min. on moist filter paper to reduce imbibitional damage after cryopreservation. Thawed buds were rehydrated for 2–4 h in sterile moist moss grass to reduce imbibitional injury. In our study, high survival was obtained with rehydration in moist moss grass in compared to a lack of rehydration (Table 2). Apparently, slow thawing for stepwise freezing and fast thawing for fast frozen samples are the optimal methods for optimizing recovery. Similarly, recovery conditions including dark incubation and rehydration in sterile moist moss grass for different durations led to higher survival in dormant buds of almond [32] and Himalyan mulberry [33].
Conventional methods of propagation of mulberry through cuttings and grafting have certain limitations. Bapet et al. [34] reported that only 30–40% of stem cuttings survive the time period between pruning, transportation, and final transplantation. Ohyama and Oka [35] found that rooting from stem cutting depends on the environmental factors and physiological state of the cuttings, while grafting depends on the internal factors such as compatibility, nutrient and moisture content of the scion, activity of the cambium, as well as external factors such as atmospheric temperature and soil mixture [36]. In vitro propagation has been utilized for large-scale propagation of several tree species [37,38,39,40,41]. The regeneration of complete plantlets in vitro from apical/axillary shoot buds or nodal explants [35,42,43,44] and from cryostored buds [15,29,45] have been reported in several mulberry species. According to Ivanicka [43], MS medium [46] is the most efficient for mulberry species. In the present study, shoot formation was observed faster in case of 0.1 mgL−1 TDZ but survival percent was found to be at its maximum in case of 1 mgL−1 BAP. Similarly, thidiazuron (TDZ) at 0.1 mgL–1 or benzylaminopurine (BAP) at 1.0 mg L–1, added separately, gave the best rates of shoot initiation and shoot induction, and required less time for bud sprouting in M. alba, M. indica, and M. laevigata [47].
Low survival and shoot formation in Morus laevigata, M. serrata, M. tilliaefolia, M. rubra, M. rotundiloba, and M. cathayana showed a need for modification of culturing conditions [48]. In the present study, M. cathayana, M. rubra, and M. serrata struggled to survive. Khurana et al. [49] found that MS medium supplemented with BAP (1 mgL−1) and low concentration of GA3 (0.1 mgL−1) and NAA (0.2 to 0.5 mgL−1) increased shoot elongation after cryopreservation.
Exposure of tissues to light during early recovery after liquid nitrogen exposure increased damage in shoot apices of Solanum tuberosum [50]. Because freezing and desiccation stresses are associated with cryostorage, tissues exposed to liquid nitrogen may be predisposed to other stresses, such as photo oxidative stress, during thawing and recovery [51,52]. Elstner et al. [53] found when plant tissues are exposed to stress, susceptibility to photo oxidative damage is increased. Based on the hypothesis that cryoexposure of tissues predisposes them to free radical attack, it is proposed that post-thaw conditions can influence oxidative damage and further reduce survival. In our study, higher survival was achieved with dark incubation. Buds that did not show any recovery on direct culturing under light conditions showed viability of 12–100% (Table 4). Overall, the current study showed that for Morus species, no significant decrement of survival was observed after 6 years of cryostorage at −196 °C at National Cryogenebank, NBPGR, New Delhi (India). Niino et al. [10] also demonstrated that no significant decrease was observed after 5 years of storage at −135 °C. Similarly, Rao et al. [14,15] also reported that the viability percentage of different Morus species was retained after 3 years of cryopreservation and was not altered.

4. Materials and Methods

Twigs about 50 cm long of mulberry of specified germplasm were collected from the field genebank of CSGRC, Hosur, Tamil Nadu, during the months of December, January, and February, wrapped in cotton bags, and air lifted to cryolab at NBPGR. Winter dormant buds were harvested from 1-year-old lateral shoots of mature trees. Winter buds were cryopreserved in liquid nitrogen using partial desiccation followed by a two-step freezing protocol, as described earlier [15,19,20,32,33].
Seven variables were tested for optimization of recovery growth.
Twigs harvested at regular intervals, during 2nd week of December to 2nd week of February, from a field genebank were examined for their moisture contents. Buds, after removing 5–7 outer scales, were pre-desiccated for periods ranging from 4–7 h over silica gel at room temperature. The moisture content (MC %) of fresh and desiccated dormant buds was determined via drying at 103 ± 2 °C in an oven for 17 h and expressed on a fresh weight basis. Dormant buds pre-desiccated to varying moisture contents were subjected to slow or fast freezing after being enclosed in 1.8 mL cryovials. The slow stepwise freezing was achieved by sequentially lowering the temperature by −5 °C/day using deep freezers up to a terminal temperature of −30 °C before plunging in liquid nitrogen at −196 °C. Fast freezing was achieved by direct plunging in liquid nitrogen.
Thawing was achieved using 2 methods: slowly, by keeping LN-retrieved cryovials in air at ambient temperature for about 40 min; or fast, by plunging in a water bath maintained at 38 °C for 3–5 min. Differently treated buds before culturing in vitro were rehydrated by sterile moist moss grass for 2 h at room temperature and the results compared with those cultured without rehydration. The retrieved buds were placed in vitro after further removal of 2 outer scales and washing with Tween20 for 15 min, followed by continuous water washing for 10 min. Buds were then surface sterilized with sodium hypochloride for 9 min and rinsed repeatedly three times with sterile autoclaved distilled water (5 min. each). The sterilized buds were then cultured on MS medium with 3% sucrose (w/v) and solidified with 0.8% agar. MS medium was initially supplemented with 1 mgL−1 6-benzylaminopurine (BAP) and 0.1 mgL−1 thidiazuron (TDZ) for bud sprouting. After bud sprouting, they were subcultured in the MS medium supplemented with 1 mgL−1 BAP and 0.1 mgL−1 GA3 for elongation.
The in vitro cultured buds were incubated under complete darkness in a culture room at 25 ± 2 °C for 7 days. Tubes were then transferred under dim light by remaining on upper racks/shelves of the culture trolley for 3 days. Cultures were finally transferred to normal light conditions of 65 ± 10 µmol m−2 s−1 with a 16 h photoperiod in in vitro culture room.

Statistical Analysis

A total of 25 explants were used in all the experiments with three replications, and the mean values were considered. Standard errors (SE) of the arithmetic means were calculated for each experiment. The data were analyzed by analysis of variance (ANOVA) in SPSS software for Windows (Release 15.0; SPSS Inc., Chicago, IL, USA). Significant differences between means were assessed using Duncan’s multiple range test (DMRT) at p ≤ 0.05.

5. Conclusions

The present study is the first report of physiological studies and cryostorage of mulberry dormant buds harvested from plants grown in subtropical conditions where temperatures do not fall below 12 °C in any season. The different factors were investigated to influence recovery conditions. In temperate species of apple, pear, etc., the buds have been recorded to be harvested only after plants have been exposed to subzero temperatures for a minimum of 72 h. In this study, in which dormant buds have been harvested from subtropical zone, there is no such consideration. Hence, moisture levels in buds for three different winter months, namely December, January, and February, when leaves were shed, were used as criteria. During acclimatization, buds are reported to lose their water content to different extents. In our study, the buds harvested in February showed the lowest moisture content values in comparison to those harvested earlier or later, and hence were found to be optimal. Although mulberry plants grown at CSGRC, Hosur, are not exposed to any cold in nature, there is a decline in moisture content in a certain growth period which can be equated to maximum dormancy or cold acclimation, although not fully.

Author Contributions

Conceptualization, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); methodology R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); software, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); validation, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); formal analysis, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); investigation, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); data curation, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); writing—original draft preparation, R.C. (Ravish Choudhary), S.K.M. and R.C. (Rekha Chaudhury); writing—review and editing, R.C. (Ravish Choudhary), S.K.M., A.A.R. and R.C. (Rekha Chaudhury); visualization, S.K.M. and R.C. (Rekha Chaudhury); supervision, S.K.M. and R.C. (Rekha Chaudhury). All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

Not applicable.

Acknowledgments

The authors are grateful to Director, NBPGR, New Delhi for providing laboratory facilities, Central Sericultural Germplasm Resources Centre, Hosur, Tamil Nadu for providing experimental samples and Central Silk Board (CSB), Govt. of India, Thally road, Hosur, Tamil Nadu for providing financial support.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Nadeem, M.A.; Habyarimana, E.; Çiftçi, V.; Nawaz, M.A.; Karaköy, T.; Comertpay, G.; Shahid, M.Q.; Hatipoğlu, R.; Yeken, M.Z.; Ali, F.; et al. Characterization of genetic diversity in Turkish common bean gene pool using phenotypic and whole-genome DArTseq-generated silicoDArT marker information. PLoS ONE 2018, 13, e0205363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Grygorieva, O.; Klymenko, S.; Kuklina, A.; Vinogradova, Y.; Vergun, O.; Sedlackova, V.H.; Brindza, J. Evaluation of Lonicera caerulea L. genotypes based on morphological characteristics of fruits germplasm collection. Turk. J. Agric. For. 2021, 45, 850–860. [Google Scholar] [CrossRef]
  3. Juric, S.; Vlahovicek-Kahlina, K.; Duralija, B.; Maslov Bandic, L.; Nekic, P.; Vincekovic, M. Stimulation of plant secondary metabolites synthesis in soilless cultivated strawberries (Fragaria × ananassa Duchesne) using zinc-alginate microparticles. Turk. J. Agric. For. 2021, 45, 324–334. [Google Scholar] [CrossRef]
  4. Ercisli, S.; Orhan, E. Some physico-chemical characteristics of black mulberry (Morus nigra L.) genotypes from Northeast Anatolia region of Turkey. Sci. Hortic. 2008, 116, 41–46. [Google Scholar] [CrossRef]
  5. Gecer, M.K.; Akin, M.; Gundogdu, M.; Eyduran, S.P.; Ercisli, S.; Eyduran, E. Organic acids, sugars, phenolic compounds, and some horticultural characteristics of black and white mulberry accessions from Eastern Anatolia. Can. J. Plant Sci. 2016, 96, 27–33. [Google Scholar] [CrossRef] [Green Version]
  6. Kostic, D.A.; Dimitrijevic, D.S.; Mitic, S.S.; Mitic, M.N.; Stojanovic, G.S.; Zivanovic, A. Phenolic content and antioxidant activities of fruit extracts of Morus nigra L. (Moraceae) from Southeast Serbia. Trop. J. Pharm. Res. 2013, 12, 105–110. [Google Scholar]
  7. Sanjappa, M. Genetic resources of mulberry and utilization. In Geographical Distribution and Exploration of the Genus Morus L. (Moraceae); Sengupta, K., Dandin, S.B., Eds.; CSRTI: Mysore, India, 1989; pp. 4–7. [Google Scholar]
  8. Hooker, J.D. The Flora of British India; Secretary of State for India Council: London, UK, 1885; Volume IV, p. 185. [Google Scholar]
  9. Tikader, A.; Vijayan, K.; Kamble, C.K. Conservation and management of mulberry germplasm through biomolecular approaches—A review. Biotech. Mol. Biol. Rev. 2009, 4, 92–104. [Google Scholar]
  10. Niino, T.; Shirata, K.; Oka, S. Viability of mulberry winter buds cryopreserved for 5 years at −135 °C. J. Sericul. Sci. Jpn. 1995, 64, 370–374. [Google Scholar]
  11. Engelmann, F. Importance of cryopreservation for the conservation of plant genetic resources. In Cryopreservation of Tropical Plant Germplasm- Current Research Progress and Applications; Engelmann, F., Takagi, H., Eds.; JIRCAS: Tsukuba, Japan; IPGRI: Rome, Italy, 2000; pp. 8–20. [Google Scholar]
  12. Chaudhury, R.; Malik, S.K. Genetic conservation of plantation crops and spices using cryopreservation. Ind. J. Biotechnol. 2004, 3, 348–358. [Google Scholar]
  13. Towill, L.E.; Ellis, D.D. Cryopreservation of dormant buds. In Plant Cryopreservation. A Practical Guide; Reed, B.M., Ed.; Springer: New York, NY, USA, 2008; pp. 421–442. [Google Scholar]
  14. Rao, A.A.; Chaudhury, R.; Kumar, S.; Velu, D.; Saraswat, R.P.; Kamble, C.K. Cryopreservation of mulberry germplasm core collection and assessment of genetic stability through ISSR markers. Int. J. Ind. Entomol. 2007, 15, 23–33. [Google Scholar]
  15. Rao, A.A.; Chaudhury, C.; Malik, S.K.; Kumar, S.; Ramachandran, R.; Qadri, S.M.H. Mulberry biodiversity conservation through cryopreservation. Vitro Cell. Develop. Biol. Plant 2009, 45, 639–649. [Google Scholar]
  16. Sakai, A.; Nishiyama, Y. Cryopreservation of winter vegetative buds of hardy fruit trees in liquid nitrogen. HortScience 1978, 13, 225–227. [Google Scholar] [CrossRef]
  17. Niino, T. Conservation of Plant Genetic Resources In Vitro; Razdan, M.K., Cocking, E.C., Eds.; Science Publishers: New York, NY, USA, 2000; pp. 195–224. [Google Scholar]
  18. Sakai, A. Cryopreservation of germplasm of woody plants. In Biotechnology in Agriculture and Forestry, Cryopreservation of Plant Germplasm; Bajaj, Y.P.S., Ed.; Springer: Berlin/Heidelberg, Germany, 1995; Volume 32, pp. 53–69. [Google Scholar]
  19. Choudhary, R.; Malik, S.K.; Chaudhury, R.; Pal, D.; Patil, P.; Sharma, K.C. Scanning electron microscopic study on freezing behaviors of tissue cells in dormant bud of mulberry (Morus sp). Bangladesh J. Bot. 2015, 44, 383–387. [Google Scholar]
  20. Choudhary, R.; Malik, S.K.; Chaudhury, R.; Pal, D.; Patil, P.; Ercisli, S.; Sharma, K.C. Study of cryopreservation and freezing behavior in almond winter bud. Erwerbs-Obstbau 2022, 64, 507–511. [Google Scholar] [CrossRef]
  21. Tyler, N.J.; Stushnoff, C. Dehydration of dormant apple buds at different stages of cold acclimation to induce cryopreservability in different cultivars. Can. J. Plant Sci. 1988, 68, 1169–1176. [Google Scholar] [CrossRef] [Green Version]
  22. Stushnoff, C. Cryopreservation of apple genetic resources. Can. J. Plant Sci. 1987, 67, 1151–1154. [Google Scholar] [CrossRef] [Green Version]
  23. Tyler, N.J.; Stushnoff, C.; Gusta, L.V. Freezing of water in dormant vegetative apple buds in relation to cryopreservation. Plant Physiol. 1988, 87, 201–205. [Google Scholar] [CrossRef]
  24. Bilavcik, A.; Faltus, M.; Zamecnik, J. The survival of pear dormant buds at ultra-low temperatures. Plants 2021, 10, 2502. [Google Scholar] [CrossRef]
  25. Niino, T.; Shirata, K.; Sakai, A. Five-year storage of winter mulberry buds (376 cultivars) at −135 °C. Crybiology 1994, 31, 605. [Google Scholar]
  26. Forsline, P.L.; McFerson, J.R.; Lamboy, W.F.; Towill, L.E. Development of base and active collections of Malus germplasm with cryopreserved dormant buds. Acta Hortic. 1999, 484, 75–78. [Google Scholar] [CrossRef]
  27. Suzuki, M.; Niino, T.; Akihama, T.; Oka, S. Shoot formation and plant regeneration of vegetative pear buds cryopreserved at −150 °C. J. Jpn. Soc. Hort. Sci. 1997, 66, 29–34. [Google Scholar] [CrossRef]
  28. Jenderek, M.M.; Forsline, P.; Postman, J.; Stover, E.; Ellis, D. Effect of geographical location, year, and cultivar on survival of Malus sp. dormant buds stored in vapors of liquid nitrogen. Hortscience 2011, 46, 1230–1234. [Google Scholar] [CrossRef] [Green Version]
  29. Yakuwa, H.; Oka, S. Plant regeneration through meristem culture from vegetative buds of mulberry (Morus bombycis koidz.) stored in liquid nitrogen. Ann. Bot. 1988, 62, 79–82. [Google Scholar] [CrossRef]
  30. Niino, T.; Sakai, A.; Enomoto, S.; Magoshi, J.; Kato, S. Cryopreservation of in vitro-grown shoot tips of mulberry by vitrification. Cryoletters 1992, 13, 303–312. [Google Scholar]
  31. Touchell, D.; Walters, C. Recovery of embryos of Zizania palustris following exposure to liquid nitrogen. Cryoletters 2000, 21, 261–270. [Google Scholar]
  32. Choudhary, R.; Malik, S.K.; Chaudhury, R.; Sharma, K.C. Long-term conservation of dormant buds of Prunus dulcis (Miller) D.A. Webb. Using three different new cryotechniques. Rom. Biotechnol. Lett. 2014, 19, 9583–9592. [Google Scholar]
  33. Choudhary, R.; Chaudhury, R.; Malik, S.K. Successful cryoconservation of dormant buds of Himalyan mulberry (Morus laevigata Wall. ex Brandis) using three different cryotechniques. Ind. J. Exp. Biol. 2018, 56, 342–350. [Google Scholar]
  34. Bapet, V.A.; Mhatre, M.; Rao, P.S. Propagation of Morus indica L. (Mulberry) by encapsulated shoot buds. Plant Cell Rep. 1987, 6, 393–395. [Google Scholar] [CrossRef]
  35. Ohyama, K.; Oka, S. Mulberry. In Cell and Tissue Culture in Forestry; Bonga, J.M., Durzan, D.J., Eds.; Martinus-Nijhoff: The Hague, The Netherlands, 1987; Volume 3, pp. 272–284. [Google Scholar]
  36. Rajan, M.V.; Ravindran, S. Propagation method of mulberry germplasm under tropical conditions. In Genetic Resources of Mulberry and Utilization; Sengupta, K., Dandin, S.B., Eds.; CSRTI: Mysore, India, 1989; pp. 81–86. [Google Scholar]
  37. Hutchinson, J.F. Tissue culture propagation of fruit trees. In Proceeding Symposium on Tissue Culture of Economically Important Plants; Rao, A.N., Ed.; COSTED/ANBS: Singapore, 1981; pp. 113–120. [Google Scholar]
  38. Litz, R.E.; Moore, G.A.; Srinivasan, C. In vitro systems for propagation and improvement of tropical fruits and palms. Hortic. Rev. 1985, 7, 157–200. [Google Scholar]
  39. Bajaj, Y.P.S. Biotechnology of tree improvement for rapid propagation and biomass energy production. In Biotechnology in Agriculture and Forestry; Bajaj, Y.P.S., Ed.; Springer: Berlin/Heidelberg, Germany, 1986; Volume 1, pp. 1–23. [Google Scholar]
  40. Zimmerman, R.H. Propagation of fruit, nut, and vegetable crops-overview. In Tissue Culture as a Plant Production System for Horticultural Crops; Zimmerman, R.H., Griesbach, R.J., Hammerschlag, F.A., Lawson, R.H., Eds.; Martinus Nijhoff: Dordrecht, The Netherlands, 1986; pp. 183–200. [Google Scholar]
  41. Hammerschlag, F.A. Tissue culture as a plant production system for horticultural crops. In Temperate Fruits and Nuts; Zimmerman, R.H., Griesbach, R.J., Hammerschlag, F.A., Lawson, R.H., Eds.; Martnus Nijhoff: Dordrecht, The Netherlands, 1986; pp. 221–236. [Google Scholar]
  42. Mhatre, M.; Bapat, V.A.; Rao, P.S. Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.). Plant Cell Rep. 1985, 4, 78–80. [Google Scholar] [CrossRef]
  43. Ivanicka, J. In vitro micropropagation of mulberry, Morus nigra L. Sci. Hort. 1987, 32, 33–40. [Google Scholar] [CrossRef]
  44. Hossain, M.; Rahman, S.M.; Zaman, A.; Joarder, O.I.; Islam, R. Micropropagation of Morus laevigata Wall. from mature trees. Plant Cell Rep. 1992, 11, 522–524. [Google Scholar] [CrossRef]
  45. Chaudhury, R.; Rao, A.A.; Malik, S.K.; Kumar, S.; Narayanamurthy, T.; Thangavelu, K. Conservation of mulberry germplasm using cryotechnology. In Proceedings of the 20th Congress of the International Sericulture Commission, Central Silk Board, Bangalore, India, 15–18 December 2005; pp. 4–9. [Google Scholar]
  46. Murashige, T.; Skoog, F. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant 1962, 15, 473–497. [Google Scholar] [CrossRef]
  47. Choudhary, R.; Chaudhury, R.; Malik, S.K. Development of an efficient regeneration and rapid clonal multiplication protocol for three different Morus species using dormant buds as explants. J. Hort. Sci. Biotechnol. 2015, 90, 245–253. [Google Scholar] [CrossRef]
  48. Pattnaik, S.K.; Chand, P.K. Rapid clonal propagation of three mulberries, Morus cathayana Hemsl., M. ihou Koiz. and M. serrata Roxb., through in vitro culture of apical shoot buds and nodal explants from mature trees. Plant Cell Rep. 1997, 16, 503–508. [Google Scholar] [PubMed]
  49. Khurana, P.; Bhatnagar, S.; Kumari, S. Tissue culture and micro propagation studies in mulberry: An overview. Ind. J. Sericul. 2003, 422, 93–100. [Google Scholar]
  50. Benson, E.E.; Harding, K.; Smith, H. The effects of pre-and post-freeze light on the recovery of cryopreserved shoot-tips of Solanum tuberosum. Cryoletters 1989, 10, 323–344. [Google Scholar]
  51. Benson, E.E. Free Radical Damage in Stored Plant Germplasm; IBPGR: Rome, Italy, 1990. [Google Scholar]
  52. Benson, E.E.; Withers, L.A. Gas chromatographic analysis of volatile hydrocarbon production by cryopreserved plant tissue cultures: A non-destructive method for assessing stability. Cryoletters 1987, 8, 35–46. [Google Scholar]
  53. Elstner, E.F.; Wagner, G.A.; Schutz, W. Activated oxygen in green plants in relation to stress situations. Curr. Top. Plant Biochem. Physiol. 1988, 7, 159–187. [Google Scholar]
Plants 12 00225 g001 550
Figure 1. Moisture content (MC%) of dormant buds of different mulberry germplasm harvested in three winter months. MC% values are cumulative of 3 years data. Error bars showing mean values ±SD (n = 3).
Figure 1. Moisture content (MC%) of dormant buds of different mulberry germplasm harvested in three winter months. MC% values are cumulative of 3 years data. Error bars showing mean values ±SD (n = 3).
Plants 12 00225 g001
Plants 12 00225 g002 550
Figure 2. Viability of dormant buds of different Morus species after cryostorage. Error bars showing mean values ± SD (n = 3). (IMC = initial moisture content; DMC = moisture content after desiccation; DC = desiccation; AC = after cryopreservation).
Figure 2. Viability of dormant buds of different Morus species after cryostorage. Error bars showing mean values ± SD (n = 3). (IMC = initial moisture content; DMC = moisture content after desiccation; DC = desiccation; AC = after cryopreservation).
Plants 12 00225 g002
Plants 12 00225 g003 550
Figure 3. Relationship between moisture content% and viability% after slow freezing (SF) and fast freezing (FF) using fast thawing in M. indica. Error bars showing mean values ±SD (n = 3).
Figure 3. Relationship between moisture content% and viability% after slow freezing (SF) and fast freezing (FF) using fast thawing in M. indica. Error bars showing mean values ±SD (n = 3).
Plants 12 00225 g003
Plants 12 00225 g004 550
Figure 4. Recovery growth of mulberry from cryopreserved dormant buds after using different critical factors. (A) Mulberry tree in field genebank in winter period, (B) explants as a twig or dormant buds for cryopreservation, (C) freezing at different temperature and finally plunged in cryotank, (D) rehydration in sterile moist moss grass for different duration, (E) in vitro recovery of cryopreserved dormant buds of different Morus species, in vitro sprouting of dormant buds after cryostorage, (F) shoot multiplication and elongation, (G) rooting, (H) hardening of in vitro recovered plant. All photos were taken by the authors and all photos are original.
Figure 4. Recovery growth of mulberry from cryopreserved dormant buds after using different critical factors. (A) Mulberry tree in field genebank in winter period, (B) explants as a twig or dormant buds for cryopreservation, (C) freezing at different temperature and finally plunged in cryotank, (D) rehydration in sterile moist moss grass for different duration, (E) in vitro recovery of cryopreserved dormant buds of different Morus species, in vitro sprouting of dormant buds after cryostorage, (F) shoot multiplication and elongation, (G) rooting, (H) hardening of in vitro recovered plant. All photos were taken by the authors and all photos are original.
Plants 12 00225 g004
Table
Table 1. Viability and dormancy status of dormant buds of different Morus species.
Table 1. Viability and dormancy status of dormant buds of different Morus species.
S. NoSpeciesAccession No.Initial Moisture Content (%) Initial Viability (%) Desiccated Moisture Content (%)
(DMC) 		Viability (%) at DMCViability (%) after Cryo-ExposureStatus
1.Morus albaEC 49382251.2 (±0.55) b90.0 (±4.08) b16.1 (±0.42) c80.0 (±2.36) b80.0 (±2.36) bSD
2.Morus australisEC 49375828.1 (±0.44) e70.0 (±4.08) c14.8 (±0.41) e30.0 (±4.71) g32.0 (±2.72) eSD
3.Morus bombycisEC 49378540.7 (±0.47) cd80.0 (±2.36) bc13.3 (±0.38) f70.0 (±2.36) c66.5 (±3.60) cD
4.Morus cathayanaEC 49377557.5 (±0.47) ab46.7 (±2.72) f13.8 (±0.48) f36.7 (±2.72) g30.0 (±2.72) eSD
5.Morus indicaIC 31369752.4 (±0.47) b60.0 (±2.36) d15.3 (±0.04) d45.0 (±2.36) e40.0 (±2.36) dND
6.Morus indicaIC 31370345.5 (±0.56) c95.0 (±0.00) ab15.3 (±0.51) d80.0 (±2.36) b95.0 (±2.36) abND
7.Morus indicaIC 31371151.8 (±0.24) b65.0 (±2.36) d15.6 (±0.28) d50.0 (±4.08) e40.0 (±4.71) dND
8.Morus indicaIC 31374159.6 (±0.32) a60.0 (±2.36) d17.4 (±0.01) b40.0 (±4.71) f35.0 (±4.08) eND
9.Morus indicaIC 31384351.5 (±0.65) b90.0 (±2.36) b17.8 (±0.18) b80.0 (±4.08) b80.0 (±4.71) bND
10.Morus indica × albaIC 31399256.9 (±0.61) ab100.0 (±0.00) a17.3 (±0.43) b100.0 (±0.00) a100.0 (±0.00) aND
11.Morus indica × albaIC 31383649.5 (±0.26) bc70.0 (±4.71) c16.6 (±0.14) c60.0 (±4.71) d40.0 (±4.08) dND
12.Morus laevigataIC 31378956.8 (±0.69) ab70.0 (±4.08) c15.3 (±0.32) d50.0 (±2.36) e40.0 (±4.08) dD
13.Morus latifoliaEC 49381740.2 (±0.11) cd100.0 (±0.00) a23.6 (±0.37) a80.0 (±4.71) b39.7 (±4.95) dSD
14.Morus latifoliaEC 49382351.6 (±0.47) b70.0 (±4.71) c14.9 (±0.09) e46.7 (±5.44) e40.0 (±4.71) dSD
15.Morus latifoliaEC 49383136.7 (±0.48) d100.0 (±0.00) a09.5 (±0.15) g25.0 (±0.91) h28.6 (±1.36) efD
16.Morus multicaulisEC 49376343.7 (±0.74) c70.0 (±4.08) c16.3 (±0.01) c55.0 (±2.36) de38.4 (±3.60) dSD
17.Morus rubraEC 49398837.6 (±0.54) d50.0 (±4.71) e15.5 (±0.05) d30.0 (±2.36) g25.0 (±2.36) fD
18.Morus serrataIC 31416745.4 (±0.47) c50.0 (±4.71) e15.4 (±0.01) d40.0 (±4.71) f32.0 (±2.72) eD
19.Morus sinensisIC 31397445.5 (±0.52) c80.0 (±4.71) bc14.5 (±0.24) e70.0 (±2.36) c75.0 (±2.36) bcND
Average47.5 (±0.48) 75.1 (±2.91) 15.2 (±0.24) 51.5 (±2.73) 50.4 (±3.15)
ND—non-dormant; SD—semi-dormant; D—dormant. All the values are the average ± SD of three replicates (n = 3), and the means with the same letter (superscript) in the columns are not significantly different (p < 0.05)—(Duncan’s multiple range test).
Table
Table 2. Recovery percentage for dormant buds of different Morus species cryoconserved using slow and fast freezing after partial desiccation with and without rehydration.
Table 2. Recovery percentage for dormant buds of different Morus species cryoconserved using slow and fast freezing after partial desiccation with and without rehydration.
Accession NoRecovery Percentage (%)
Slow Freezing (2-Step Freezing) Fast Freezing (Direct Plunging in LN)
Slow Thawing with RehydrationSlow Thawing without RehydrationFast Thawing with RehydrationFast Thawing without RehydrationSlow Thawing with RehydrationSlow Thawing without RehydrationFast Thawing with RehydrationFast Thawing without Rehydration
M. indica × alba
[IC 493875]		63.3 (±2.72) c50.0 (±4.71) c41.7 (±4.91) b25.0 (±2.36) b36.4 (±2.72) a12.5 (±1.18) ab25.3 (±0.47) a16.7 (±0.33) a
M. alba
[IC 313736]		80.0 (±2.36) b75.0 (±2.36) a07.7 (±0.14) c00.0 (±0.00) 14.3 (±0.01) b10.2 (±0.42) b06.7 (±0.48) c00.0 (±0.00)
M. indica
[IC 313977]		90.0 (±4.71) a66.7 (±2.72) b52.4 (±0.48) a33.3 (±2.72) a11.1 (±0.47) b03.3 (±0.27) c13.1 (±1.36) b06.7 (±0.27) b
M. indica
[IC 313887]		90.9 (±4.09) a50.0 (±4.71) c41.7 (±1.36) b16.7 (±2.72) c33.3 (±1.22) a16.7 (±2.72) a20.0 (±4.71) ab06.7 (±0.27) b
Average81.1 (±3.47)60.4 (±4.83)35.8 (±1.72)18.7 (±1.95)23.8 (±1.10)10.7 (±1.15)16.3 (±1.70)7.5 (±0.22)
All the values are the average of three replicates (n = 3), and the mean with the same letter (superscript) in the columns are not significantly different (p < 0.05)—(Duncan’s multiple range test).
Table
Table 3. Effect of different plant growth regulators (PGRs) on viability percentage of dormant buds of different Morus species.
Table 3. Effect of different plant growth regulators (PGRs) on viability percentage of dormant buds of different Morus species.
PGRM. alba
IC 314257		M. bombycis
IC 405829		M. indica
IC 314248		M. indica
IC 405817		M. indica
IC 314247		M. indica
IC 314094		M. indica
IC 405800		M. indica
IC 314255		Avg.
Viability (%)
BAP (1 mgL−1) 33.3 (±2.72) a73.3 (±2.72) b73.3 (±5.44) a83.3 (±6.80) a93.3 (±2.72) a70.0 (±4.71) a86.7 (±5.44) b100.0 (±0.00) 76.6
TDZ (0.1 mgL−1) 26.7 (±2.27) b96.7 (±2.72) a66.7 (±2.72) b73.3 (±2.72) b86.7 (±5.44) b63.3 (±2.72) b90.0 (±4.71) a100.0 (±0.00) 75.4
All the values are the average of three replicates (n = 3), and the mean with the same letter (superscript) in the columns are not significantly different (p < 0.05)—(Duncan’s multiple range test).
Table
Table 4. Improved viability of cryostored Morus species after rehydration and culturing in the dark.
Table 4. Improved viability of cryostored Morus species after rehydration and culturing in the dark.
S.
No		SpeciesAccession No.Viability (%)
Direct Culturing *After Modifying Culture Conditions **
1Morus albaEC 4937990.0012.0 (±1.63) d
2Morus bombycisEC 4938210.0020.0 (±4.71) cd
3Morus cathayanaEC 4937750.0030.0 (±4.71) c
4Morus latifoliaEC 4937790.00100.0 (±0.00) a
5Morus latifoliaEC 4938190.0075.0 (±2.36) b
6Morus sinensisIC 3139740.0075.0 (±2.36) b
* Buds descaled and inoculated in vitro; ** buds descaled then rehydrated in 100% RH for 2 h and then cultured for 7 days in complete dark and 4 days in dim light. All the values are the average, ± SD of three replicates (n = 3), and the means with the same letter (superscript) in the columns are not significantly different (p < 0.05)—(Duncan’s multiple range test).
Table
Table 5. Viability of mulberry germplasm after different duration of cryostorage.
Table 5. Viability of mulberry germplasm after different duration of cryostorage.
S.
No		SpeciesAccession
No.		Viability (%) after Different
Duration of Cryostorage
(3 Months) (6 Years)
1Morus albaIC 31370330.0 (±5.68) 30.0 (±4.71)
2Morus bombycisEC 49378566.7 (±2.72) 60.0 (±4.71)
3Morus indicaIC 31370395.0 (±2.36) 90.0 (±2.36)
4Morus indicaIC 31370340.0 (±4.71) 40.0 (±4.71)
5Morus indicaIC 31370390.0 (±4.71) 80.0 (±4.08)
6Morus indicaIC 31370355.0 (±2.36) 53.3 (±2.72)
7Morus indica × albaIC 31370360.0 (±2.72) 60.0 (±2.72)
8Morus rubraEC 49398825.0 (±2.36) 20.0 (±4.71)
Average57.7 (±3.45) 54.2 (±3.84)
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Choudhary, R.; Malik, S.K.; Chaudhury, R.; Rao, A.A. Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm. Plants 2023, 12, 225. https://doi.org/10.3390/plants12020225

AMA Style

Choudhary R, Malik SK, Chaudhury R, Rao AA. Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm. Plants. 2023; 12(2):225. https://doi.org/10.3390/plants12020225

Chicago/Turabian Style

Choudhary, Ravish, Surendra Kumar Malik, Rekha Chaudhury, and Atmakuri Ananda Rao. 2023. "Optimized Recovery of Cryostored Dormant Buds of Mulberry Germplasm" Plants 12, no. 2: 225. https://doi.org/10.3390/plants12020225

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop