In Vitro Preservation of Somatic Seeds and Nonencapsulated Hemp Shoot Tips ^†

Abstract

Synthetic seed technology and cold storage methods provide genetic uniformity, paving the way for pest- and disease-free plants that are easy to handle. The aim of this study was to develop protocols for the cold storage of nonencapsulated and alginate-capsulated explants of Cannabis sativa L. Axillary shoot tips derived from in vitro-grown plants that were used as explants and stored for up to 9 months at 4 °C in the dark. Somatic seeds were produced in 3% sodium alginate and Murashige and Skoog (MS) medium salt and stored for up to 3 months. After 6 months of cold storage, the highest regrowth of 45% was recorded for the nonencapsulated explants. The recovery of somatic seeds was 90% under the same storage condition after 3 months. Well-developed, regenerated plants from encapsulated explants successfully acclimatized.

1. Introduction

Synthetic seed technology and cold storage are used for the rapid clonal propagation of plants and germplasm preservation. These methods provides genetic uniformity, paving the way for pest- and disease-free plants that are easy to handle and transport. Various explants, such as shoot tips, nodal segments, axillary buds, somatic embryos, as well as other vegetative parts of the plant, can be encapsulated in an artificial hydrogel. The encapsulation of nonembryonic vegetative propagules has been used as a suitable alternative for micropropagation and the short-term storage of valuable medicinal plants [1,2]. Synthetic seed technology has been also used for germplasm conservation and multiplication. The choice of the initial explants and encapsulating agent and matrix, the addition of growth regulators and nutrients to the capsules, and the choice of experimental conditions substantially influence the success of synthetic seed production, as well as their storage and regeneration [3]. Prepared, encapsulated synthetic seeds can be stored at low temperatures. In vitro cold storage without regular subcultures allows one to rationalize the production of nuclear stocks and maintain gene collections [1]. Storage at low temperatures reduces metabolic rate, minimizes the risk of somaclonal variation, and prolongs storage time. However, low-temperature treatment may induce chilling stress, triggering elevated levels of reactive oxygen species (ROS) and causing damage to propagules during storage [4,5].

The cold storage protocols for Cannabis sativa L. are scare and limited to drug-type chemotypes [5,6,7,8]. In this study, we tested industrial hemp genotypes using shoot tips as initial explants. The aim of this study was to develop protocols for the cold storage of nonencapsulated shoot tips and for alginate-capsulated somatic seeds. The viability and survival rates obtained under various treatments and time periods of up to 9 months were considered to verify this treatment as an alternative solution for the large-scale propagation and germplasm conservation of valuable hemp genotypes.

2. Materials and Methods

2.1. Plant Material and Source of Explants

In vitro-grown hemp plants were used as the source of explants (shoot tips). Hemp cultivars Epsilon 68, Globa and hybrid Carmagnola × K290 (marked as 1565) were tested. Stock shoot cultures were grown for 3 weeks in Magenta GA7 boxes containing half-strength (½ MS) medium [9] with 0.5 mg L⁻¹ IAA (Indole-3-acetic acid), 3% sucrose, and 8.5% agar (BD BACTO™ Agar). Cultures were maintained at 25 ± 1 °C under a 18/6 h light/dark photoperiod with 80–120 μmol m² s⁻¹ fluorescent daylight. Shoot tips were removed from the in vitro-grown plants once they had reached 14–21 days of age and cultured for the next 2 weeks to stimulate the growth of lateral shoots, as described by Wróbel et al. [10]. After 2 weeks, shoot tips derived from axillary shoots were cut and used in the experiments.

2.2. Preparation and Storage of Nonencapsulated Explants

Shoot tips (0.5–1.0 cm, Figure 1a) were excised from axillary shoots and placed vertically in Magenta vessels containing ½ MS medium and 8.5% agar. Explants (30 per vessel) were stored at 4 °C in the laboratory fridge in dark up to 9 months. Every 3 months, 12–15 explants of each accession were set to regrow on the same medium (½ MS + IAA (0.5 mg L⁻¹), 3% sucrose, 8.5% agar) and placed in a growth chamber under the same conditions as the hemp cultures. The regrowth of explants was recorded after 3 weeks of culture. After this period, survival rate, the percentage of rooted plants, the percentage of callusing explants, and the number of roots per shoot were calculated, and the lengths of the shoots were measured (cm). The experiment was conducted in four replicates, each with 12–15 explants.

2.3. Preparation and Storage of Encapsulated Explants

The shoot tips (0.5–1.0 cm) from the axillary shoots of the rooted plants (Epsilon 68 and 1565) were excised and fully submerged in 3% alginic acid sodium salt with full-strength MS basal salt medium supplemented with 3% sucrose. Alginic acid-coated buds were dropped into 75 mM calcium chloride using an automatic pipette for 30 min incubation to induce hardening. The synthetic seeds were kept on ½ MS medium in Petri dishes sealed with parafilm. Dishes were stored in the laboratory fridge at 4 °C in the dark for up to 3 months. The same regrowth conditions and medium were used for the plant regeneration of the somatic seeds. The experiment was conducted in two replicates, each with 12–15 explants. All chemicals were purchased from Merck, except for the Bacto Agar (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and sucrose (POCH S.A., Gliwice, Poland).

2.4. Acclimatization Conditions

The regenerated plants (n = 30) were removed from the vessels and washed in autoclaved water before being placed in pots with sterilized soil (standard garden soil without additives) under glass covers and grown at 25 ± 1 °C (18/6 photoperiod, 80–120 μmol m² s⁻¹). After 1 week, the glass covers were replaced with plastic covers, and the plants were progressively exposed to the environmental humidity and then hardened for 3–4 weeks. After this period, the percentage of well-developed plants was calculated.

3. Results and Discussion

3.1. Storage and Recovery of Nonencapsulated Explants

One of the key factors affecting the success of cold storage is the choice of the initial explants. For this study, we used shoot tips excised from axillary shoots of in vitro-grown plants. The choice of explant was based on preliminary studies that showed better rooting and shoot regeneration for shoot tip explants compared to nodal explants. The differences in rooting rate of these two types of explants were significant in all tested hemp accessions (e.g., 86% vs. 30% for the hybrid 1565 cultivars or 82% vs. 49% for the Epsilon 68 cultivars). Therefore, the shoot tips were used as the initial explants in both experiments. Results regarding the effect of cold storage duration on the recovery of “naked” explants of hemp are presented in

Table 1. Results regarding the effect of cold storage duration on the rooting and shoot regeneration of unencapsulated explants of hemp.

Accession	Storage in Months	Survival (%)	Rooting Rate (%)	Mean No. of Roots per Explants (±SD)	Mean Shoot Length [cm]	Callusing Explants (%)
Epsilon 68	3	100.0	85.7	5.00 ± 3.61	3.08 ± 1.72	31.0
	6	58.1	27.9	2.18 ± 0.98	1.55 ± 1.40	41.9
	9	16.7	4.8	0.92 ± 0.17	0.56 ± 0.24	2.4
Carmagnola × K290 (1565)	3	100.0	71.4	4.45 ± 4.38	2.58 ± 1.56	40.5
	6	81.8	45.5	3.48 ± 1.95	1.92 ± 1.59	72.7
	9	52.4	28.6	2.82 ± 1.29	1.02 ± 1.40	11.9
Globa	3	90.7	48.8	2.48 ± 1.63	1.84 ± 1,43	72.1
	6	86.0	44.2	2.30 ± 1.90	1.50 ± 0.76	69.8
	9	78.6	35.7	2.21 ± 1.33	1.66 ± 1.49	73.8

.

Cold storage duration, as well as the hemp accession, had a significant impact on explant recovery and plant regeneration. After 6 months of storage, 58–86% explants survived, and 28–45.5% of them fully regenerated root systems and shoots (Table 1). After 9 months, only 5–36% of explants developed into vigorous plantlets. Moreover, the effect was genotype-dependent. Epsilon 68 explants were the most sensitive to chill stress and showed the poorest recovery rates after 6 and 9 months of storage. The differences between the hemp accessions may result from their adaptation to local climatic conditions (e.g., French cultivar Epsilon 68 was more sensitive than Ukrainian cultivar Globa). The Globa and 1565 accessions showed similar recovery rates of 46% and 44% after 6 months. It should be noted that the “naked” explants showed a similar recovery rate of 43%, which was recorded for encapsulated synthetic seeds of drug-type cannabis (MX) [8]. However, the lack of cold storage protocols and other studies on hemp makes it difficult to compare our results.

3.2. Storage and Recovery of Somatic Seeds

Studies on the encapsulation of Cannabis sativa L. explants are limited to a few papers and concern only high-yielding drug-type genotypes. For encapsulation, axillary buds [6] and nodal explants [5,7,8] were used. A 60% regrowth rate was noted for the synthetic seeds under storage at 15 °C for 24 weeks [8]. High regrowth rates of 70% and 90% were recorded for synthetic seeds from in vitro- and in vivo-derived plants by Zarei et al. [5]. In this study, encapsulated explants (nodal segments) were stored at 6 °C for 150 days. They found that the addition of acetylsalicylic acid (ASA) to the encapsulation matrix and light conditions during storage significantly improved the germination and regrowth rate of synthetic seeds. To the best of our knowledge, no somatic seed protocols for industrial hemp as initial explants have been developed, nor for shoot tips as initial explants. Results regarding the regrowth of the somatic seeds of both hemp accessions are presented in

Table 2. Regrowth rates of somatic seeds after 3 months of cold storage (recorded 3 weeks after germination).

Accession	Survival (%)	Rooting Rate (%)	Mean No. of Roots Per Explants (±SD)	Mean Shoot Length [cm]	Callusing Explants (%)
Epsilon 68	90	55.0	2.34 ± 1.19	3.37 ± 3.20	50.0
Carmagnola × K290 (1565)	100.0	90.0	2.98 ± 2.35	1.50 ± 1.12	70.0

and Figure 1b.

After 3 months of storage, relatively high regrowth rates (90% and 55%) were recorded for both hemp accessions. It is worth noting that the nonencapsulated explants of Epsilon 68 showed better rooting rates (85.7% vs. 55%); however, these results have been derived from preliminary experiments, and the study is still ongoing. The final results on a larger sample of explants will give a decisive answer with respect to which form of preservation and storage is optimal for the tested hemp genotypes. Well-rooted, regenerated plants from encapsulated explants successfully acclimatized, with a survival rate of 100%.

4. Conclusions

In this preliminary study, two forms of in vitro preservation were tested, as nonencapsulated shoot tips and sodium alginate-capsulated explants were stored at 4 °C. The results of these preliminary experiments show that both methods are potentially useful and suitable for hemp germplasm conservation. However, the protocol of cold storage should be optimized and adapted to the tested hemp genotype.