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The US Department of Agriculture-Agricultural Research Service (USDA-ARS), National Plant Germplasm System (NPGS) plant collections are a critical source of genetic diversity for breeding and selection of improved crops, including vegetatively propagated plants. Information on these collections is readily accessible to breeders and researchers on the internet from the Germplasm Resources Information Network (GRIN). The clonal collections are at risk for loss due in part to their genetic diversity that makes growing them in one location a challenge, but also because it is difficult to have duplicate collections without incurring great expense. The development of cryopreservation techniques during the last two decades provides a low maintenance form of security backup for these collections. National plant collections for vegetatively propagated crop plants and their wild relatives are maintained by the USDA-ARS, NPGS at 15 sites across the country. These sites include various combinations of field, greenhouse, screenhouse, and in vitro collections. Cryopreserved backup collections in liquid nitrogen storage were instituted in the 1990s, increased greatly in the 2000s with the advent of new techniques, and are continuing today. Collections of dormant buds of temperate trees, shoot tips of in vitro cultures of many crops, and embryonic axes of some large seeded or recalcitrant seeded plants are all part of the clonal backup storage system.

Production of virus-free plants is necessary to control viral diseases, import novel cultivars from other countries, exchange breeding materials between countries or regions and preserve plant germplasm. In vitro techniques represent the most successful approaches for virus eradication. In vitro thermotherapy-based methods, including combining thermotherapy with shoot tip culture, chemotherapy, micrografting or shoot tip cryotherapy, have been successfully established for efficient eradication of various viruses from almost all of the most economically important crops. The present study reviewed recent advances in in vitro thermotherapy-based methods for virus eradication since the twenty-first century. Mechanisms as to why thermotherapy-based methods could efficiently eradicate viruses were discussed. Finally, future prospects were proposed to direct further studies.

Key message We transferred the Tri6 gene into the elite barley GemCraft via new transformation method through shoot organogenesis and identified the rearrangements of transgenes and phenotypic variations in the transgenic plants . Despite its agronomic and economic importance, barley transformation is still very challenging for many elite varieties. In this study, we used direct shoot organogenesis to transform the elite barley cultivar GemCraft with the RNAi constructs containing Tri6 gene of Fusarium graminearum , which causes fusarium head blight (FHB). We isolated 4432 shoot tips and co-cultured these explants with Agrobacterium tumefaciens . A total of 25 independent T0 transgenic plants were generated including 15 events for which transgene-specific PCR amplicons were observed. To further determine the presence of transgenes, the T1 progenies of all 15 T0 plants were analyzed, and the expected PCR products were obtained in 10 T1 lines. Droplet digital (dd) PCR analysis revealed various copy numbers of transgenes in the transgenic plants. We determined the insertion site of transgenes using long-read sequencing data and observed the rearrangements of transgenes. We found phenotypic variations in both T1 and T2 generation plants. FHB disease was evaluated under growth chamber conditions, but no significant differences in disease severity or deoxynivalenol accumulation were observed between two Tri6 transgenic lines and the wildtype. Our results demonstrate the feasibility of the shoot tip transformation and may open the door for applying this system for genetic improvement and gene function research in other barley genotypes.

A prompt and dynamic clonal propagation system was developed for Hybanthus enneaspermus a medicinally significant plant using shoot tip as explants. Multiple shoots were induced on Murashige and Skoog (MS) medium invigorated with different levels of 6-benzylaminopurine (BAP), kinetin (Kin) and thidiazuron (TDZ) and AgNPs. The optimum concentration of BAP (1.5 mg L─1 ) with AgNPs (3.0 mg L─1) exhibited a determined response of 90.66% and created a maximum of 77.23 shoots with a maximum shoot length of 3.72 cm after 4 weeks. The vigorous shoots were embedded on an MS medium modified with optimum concentration of IBA 1.0 mg L─1 and AgNPs 1.0 mg L─1, which engendered the highest number of roots (9.65) with an average root length of 4.77 cm, with the response of 87%. Further, rooted plantlets were efficiently acclimatized in the soil. In addition, genetic homogeneity analysis was performed using RAPD and SCoT molecular marker, which revealed that the regenerated plants were genetically uniform and no detectable genetic difference was observed when compared to the mother plant. This efficient micropropagation protocol provides a new way for targeted gene editing for functional analysis of genes in Hybanthus enneaspermus.Key messageThe green synthesized AgNPs increased the regeneration frequency and there is no genetic variation among the invitro regenerated plants.

We report the successful cryopreservation of three economically important Rubus viruses: raspberry bushy dwarf virus (RBDV), black raspberry necrosis virus (BRNV), and Rubus yellow net virus (RYNV), using shoot tip cryopreservation in four raspberry cultivars. Virus-infected shoot tips (approximately 1.0 mm in length) containing 3–4 leaf primordia (LPs) were cryopreserved using the droplet-vitrification technique. In the cultivars ‘Zlatá Královna (ZK)’ and ‘Tulameen (TUM)’, over 90% of shoot tips survived, and more than 90% regenerated into whole shoots. All three viruses were successfully preserved in the cryopreserved tissues, with recovery rates varying depending on virus type and cultivar: RBDV was recovered at rates of 86% in ‘ZK’ and 87% in ‘TUM’; BRNV at 66% in ‘ZK’ and 45% in ‘TUM’; and RYNV at 96%, 94%, and 86% in ‘Fairview’, ‘Stiora’, and ‘ZK’, respectively. To investigate viral localization in shoot tips, in situ hybridization was used. RBDV and RYNV infected a broad range of meristematic tissues, including the apical dome and LPs, whereas BRNV showed a more limited distribution. Virus distribution varied not only among virus species but also across raspberry cultivars, suggesting genotype-specific patterns of virus localization. Post-cryopreservation viral activity was verified using micrografting and aphid transmission assays. RBDV, BRNV, and RYNV were all successfully transmitted to healthy plants via micrografting, indicating the preservation of viral infectivity. Furthermore, BRNV was effectively transmitted by large raspberry aphids from cryopreserved materials, confirming vector-mediated transmission capacity post-thaw. Overall, this study demonstrates that shoot tip cryopreservation via droplet-vitrification is a reliable and effective strategy for preservation of biologically active Rubus viruses. This approach offers a valuable biotechnological tool for virus maintenance in support of diagnostic, breeding, and virology research.

Enset is a perennial, multipurpose crop that is cultivated and consumed in Ethiopia. Nowadays, its traditional propagation systems face a challenge due to biotic and abiotic factors. Thus, shoot tip culture can be very advantageous for the quick multiplication of healthy plantlets to secure the conservation as well as propagation of the enset crop. Therefore, this study was designed to develop an efficient micro-propagation protocol for three popular multi-use enset genotypes by using locally available bulla and agar as gelling agents separately. The experiment was conducted in a completely randomized design with three replications in a factorial arrangement. About 1.0 cm long shoot tips were cultured on MS medium supplemented with 1 to 6 mg/l of BAP separately or in combination with IAA. It was found that the 8% (w/v) enset bulla was ideal and provided significant figures in the number and length of shoots and roots per shoot and also early initiation of shoots and roots when compared with 0.6% (w/v) agar-gelled MS media. MS medium containing 2.0–3.0 mg/l BAP was the appropriate concentration for in-vitro shoot induction and growth. The presence of 5.0 mg/l BAP alone, and 5.0 mg/l BAP in combination with 1.0 mg/l IAA was suitable for multiple shoot induction, whereas, 2.0 mg/l IBA and 1.0 mg/NAA separately were found to be the optimum concentration for root induction and development. Thus, bulla in addition to its alternative gelling potential with low cost has potential play an essential role in the rapid production and conservation of enset with desirable traits and disease-free plantlets for farmers.Key messageEnset is an important food security and multipurpose crop. Bulla, which is extracted from enset, is an alternative gelling agent that is more efficient in the in vitro propagation of three different multi-use enset genotypes when compared to agar gelled medium.

Cadaba trifoliata (Roxb.) Wight & Arn. (Capparaceae) is an occasional and endemic tree species of the Peninsular India. In this study, an effective in vitro regeneration system is reported for the first time for this tree, and the impact of growth regulators on the micro-morphometric growth modulations was assessed. Node explants were cultivated on Murashige and Skoog (MS) medium containing 3 types of cytokinins viz., N 6-benzyladenine (BA), N 6-furfuryladenine (kinetin/Kin), and N 6-( meta -hydroxybenzyl)adenine ( meta -Topolin/ m T). The MS medium with m T at 1.0 mg/L was found the most efficient cytokinin for bud break, resulting in a 90.3% organogenic response and producing 8.0 shoots/explants. Conversely, MS medium with 1.5 mg/L BA produced 4.0 shoots, and only 3.3 shoots resulted in 2.0 mg/L Kin. Additionally, a combination of m T (0.5 mg/L) and 0.25 mg/L indole-3-acetic acid (IAA) increased regeneration frequencies and a maximum of 23.0 shoots (5.4 cm length) were amplified, in contrast a combination of BA + IAA and Kin + IAA resulted in a lesser number of shoots comparatively with shoot tip necrosis. Microscopic analysis showed that the leaves developed on the optimal m T and IAA combination were structurally superior with moderately differentiated cuticle, uniform epidermal layers, and increased mesophyll density and vascular elements. The leaves from BA + IAA and Kin + IAA treatments exhibited micro-structural abnormalities. The highest rhizogenic response (95.8%) and a considerable number of roots (5.0 roots with 4.3 cm length) were regenerated from the shoots derived from m T and IAA combination on 1.25 mg/L indole-3-butyric acid (IBA). About 91.7% of the regenerated plantlets survived during ex vitro acclimatization. The study thus highlights the pivotal role of m T in stimulating the in vitro regeneration and alleviation of micro-structural and physiological disorders in cultures of C. trifoliata .

Trichosanthes cucumerina L. is a medicinal vine with great potential that can be used as a vegetable. In this study, an efficient in vitro regeneration system of T. cucumerina was established by inducing adventitious buds and controlling Shoot Tip Necrosis (STN) in the in vitro culture. The results of hormone single factor and orthogonal experiments showed that 6-BA had a significant effect on inducing adventitious buds. The best medium for bud proliferation was MS + 6-BA 3.0 mg·L− 1 + KIN 2.0 mg·L− 1 + NAA 0.5 mg·L− 1, and the proliferation capacity was 14.68. However, after three consecutive subcultures, a higher incidence of STN (78.86%) was observed. The results of the STN control experiment showed that reducing the concentration of inorganic salt, shortening the subculture interval, and increasing the Ca2+ in the medium could effectively control the occurrence of STN. However, reducing the concentration of inorganic salt and shortening the subculture interval excessively reduces the proliferation capacity significantly. Considering both the incidence of STN and proliferation capacity, the measures proposed in this study to alleviate STN were to keep the concentration of inorganic salt unchanged, adjust the subculture interval from 45 days to 40 days, and increase the Ca2+ concentration in the medium to 480 mg·L− 1. When the subculture interval was 40 d, the stem segments obtained the lowest STN incidence (8.3%) and higher shoot proliferation capacity (10.02) on the medium of MS + 3.0 mg·L− 1 6-BA + 2.0 mg·L− 1 KIN + 0.5 mg·L− 1 NAA + 360.36 mg·L− 1 Ca2+ (360 mg·L− 1 is the concentration of additional calcium to be added, and after this addition, the total calcium concentration in the MS medium was 480.48 mg·L− 1). The best rooting medium for stem segments was MS + 0.50 mg·L− 1 NAA; the rooting rate was 100%, and no STN occurred. After acclimation, the survival rate of the rooted plantlet reached 95% after 40 days in the soil matrix of coconut husk: humus soil: perlite = 2: 2: 1 (v/v). The results of this study not only reveal the mechanism of STN in T. cucumerina, but also provide technical support and a theoretical basis for its further breeding and cultivation.Key messageIn this study, an efficient and stable tissue culture rapid propagation system for Trichosanthes cucumerina L. was formed by controlling the shoot tip necrosis in vitro culture.

The presence of viral diseases poses a significant challenge to the high-quality, efficient, and sustainable production of apples. Virus eradication and the use of virus-free plants are currently the most crucial method for preventing viral diseases. Among the viruses affecting apples, apple stem grooving virus (ASGV) and apple chlorotic leaf spot virus (ACLSV) present particular challenges in efficient eradication from apples. This study investigated the effects of exogenous salicylic acid (SA) treatment on the efficient eradication of ASGV and ACLSV from apple cultivar ‘Yanfu 8’. Shoots were excised from in vitro 4-week-old stock and cultured in shoot proliferation medium supplemented with 10 μM SA combining thermotherapy with shoot tip culture or cryotherapy for ASGV and ACLSV eradication. The results showed that including of 10 μM SA in thermotherapy significantly reduced the concentrations of ASGV and ACLSV by 33% and 14% in shoots compared to thermotherapy without SA. SA treatment also improved the shoot tips survival and regrowth after combining 2 or 4 weeks of thermotherapy followed by shoot tip culture or shoot tip cryotherapy, while maintaining the higher (75–100%) of virus eradication efficiencies. Therefore, the application of SA in combination with thermotherapy followed or not by cryotherapy proves to be a promising approach for enhancing the efficiency of virus eradication in apple.Key MessageThe establishment of a salicylic acid combining thermotherapy with shoot tip culture or cryotherapy of apple shoots provided technical support for the eradication of apple viruses in apple plants.

For maize transgenic breeding, a major bottleneck is time need to produce homozygous transgenic plants by selfing of one or more additional generations. This study established an effective haploid maize callus regeneration system using haploid shoot tips of maize which enables to form homozygous transgenic insertions in a single generation. Haploid shoot tips were cultured for haploid embryonic callus induction, which was then transformed with a GFP-contained vector and generated transgenic haploid plants. We tested different seed sterilization methods, germination manners, shoot lengths, cutting patterns, medium components, light treatments in differentiation and particle bombardment distances. The results indicated that HgCl2 was better than H2O2 for decreasing seeds contamination rate. Seedlings with 2.0–6.0 cm in length was optimal for the high frequency of embryonic callus induction. Then, the best culture medium components were “MS + 2 mg/L 2,4-D + 1 mg/L 6-BA + 500 mg/L CH + 10 g/L Mannitol + 7 mg/L AgNO3 + 1 mg/L B5” and “MS + 1 mg/L 6-BA + 0.4 mg/L IBA + 500 mg/L CH + 1 mL/L Cephalothin”, respectively, for callus induction and regeneration. Callus with 4 mm in size and 4 generations of subculture was better than the others for particle bombardment to obtain positive transgenic plants. Among the six genotypes of haploids, haploid 18-599 W had the highest induction rate of embryonic callus. Interestingly, each of the haploids did not show different induction rate compared to its diploid, except haploid 18-599R. Our research provides new insights for rapid homozygosis of transformation events in maize.Key MessageAn effective haploid maize shoot tips callus regeneration system was established which enabled homozygous transgenic plants to be obtained in a single generation.