Recent Advances of Biotechnological Tools on Diverse Species of Citrus: Current Applications and Future Prospects

Based on the long term conservation of several endangered and indigenous species of Citrus, significant impact of biotechnological tools particularly in terms of in-vitro micropropagation methods in addition to synthetic seed production using encapsulation of plant propagules including shoot tips, nodal segments, androgenic embryos, embryogenic callus, etc. in sodium alginate has been highlighted in this manuscript. When seed is not available in enough quantity for raising seedlings for rootstock or have low levels of polyembryony and do not produce adequate quantities of nucellar seedlings, then micropropagation techniques could quickly supply in vitro regenerated rootstock or budwood. Rapid, mass-production and cost-effective biotechnological tools for propagation of citrus rootstocks and budwood would be of great importance in this regard. Reports on another aspect of long term conservation particularly based on storage of cells, tissues and organs of drought tolerant species of Citrus at ultra-low temperature preferably at -196 ºC via applications of cryopreservation techniques using vitrification and encapsulation or dehydration methods has been highlighted in this manuscript. In addition, several research on techniques of in-vitro micrografting using superior scion and rootstocks of two different species of Citrus with an objective of eradication of virus infected citrus stocks for successful production of grafts have been reviewed. Furthermore, effects of explants either through direct and indirect regeneration and conversion into a complete disease free plantlet using suitable synthetic nutrient media along with plant growth regulators at various concentrations and combinations have been highlighted in this manuscript. Hence, the current review is primarily focused on the applications and its effects of superior biotechnological tools for long term conservation of diverse species of citrus for further increasing the potentiality of Citrus industries in addition to genetic improvement and genetic resource conservation.

Cryopreservation of Agronomic Plant Germplasm Using Vitrification-Based Methods: An Overview of Selected Case Studies

Numerous environmental and endogenous factors affect the level of genetic diversity in natural populations. Genetic variability is the cornerstone of evolution and adaptation of species. However, currently, more and more plant species and local varieties (landraces) are on the brink of extinction due to anthropopression and climate change. Their preservation is imperative for the sake of future breeding programs. Gene banks have been created worldwide to conserve different plant species of cultural and economic importance. Many of them apply cryopreservation, a conservation method in which ultra-low temperatures (−135 °C to −196 °C) are used for long-term storage of tissue samples, with little risk of variation occurrence. Cells can be successfully cryopreserved in liquid nitrogen (LN) when the adverse effect of ice crystal formation and growth is mitigated by the removal of water and the formation of the so-called biological glass (vitrification). This state can be achieved in several ways. The involvement of key cold-regulated genes and proteins in the acquisition of cold tolerance in plant tissues may additionally improve the survival of LN-stored explants. The present review explains the importance of cryostorage in agronomy and presents an overview of the recent works accomplished with this strategy. The most widely used cryopreservation techniques, classic and modern cryoprotective agents, and some protocols applied in crops are considered to understand which parameters provide the establishment of high quality and broadly applicable cryopreservation. Attention is also focused on the issues of genetic integrity and functional genomics in plant cryobiology.

Advancing Semen Evaluation Using Lipidomics

Developing a deeper understanding of biological components of sperm is essential to improving cryopreservation techniques and reproductive technologies. To fully ascertain the functional determinants of fertility, lipidomic methods have come to the forefront. Lipidomics is the study of the lipid profile (lipidome) within a cell, tissue, or organism and provides a quantitative analysis of the lipid content in that sample. Sperm cells are composed of various lipids, each with their unique contribution to the overall function of the cell. Lipidomics has already been used to find new and exciting information regarding the fatty acid content of sperm cells from different species. While the applications of lipidomics are rapidly evolving, gaps in the knowledge base remain unresolved. Current limitations of lipidomics studies include the number of available samples to analyze and the total amount of cells within those samples needed to detect changes in the lipid profiles across different subjects. The information obtained through lipidomics research is essential to systems and cellular biology. This review provides a concise analysis of the most recent developments in lipidomic research. This scientific resource is important because these developments can be used to not only combat the reproductive challenges faced when using cryopreserved semen and artificial reproductive technologies in livestock such as cattle, but also other mammals, such as humans or endangered species.

INVESTIGATING INTERACTION OF WATER WITH PRIMARY MEMBRANES OF FISH EGGS AS SIGNAL FOR THEIR RESTRUCTURING

The article outlines studying the interaction of water with the primary membranes of fish eggs as a signal for their restructuring, which is important for developing the cryopreservation techniques. The reproductive cells of sterlet (Acipenser ruthenus Linnaeus, 1758) obtained during the spawning campaign served as the object of research. Water as a main activator of the functions of fish reproductive cells contacting with primary membranes of eggs (two yellows and one surface jelly-like) triggers a mechanism that changes the structure and functional activity of eggs, stimulates the preparation of eggs for fertilization, provoking the rearrangement of organelles and changes in the membranes. The latter are activated contacting with water and, regardless of the fact of fertilization, acquire various properties (stickiness, buoyancy, increased strength) necessary for the further development of embryos, depending on the conditions of incubation. Changes in the properties of the membranes are stipulated by the presence of certain fatty acids, further chemical transformations of which are the main factor of their changes, according to the biology of reproduction.

A durable pollination raincoat for hybridization during monsoon in coconut

Large-scale production of hybrid seed nuts of coconut is necessary to match the high demand for hybrid seedlings. But in major coconut producing states such as Kerala, Karnataka and Goa hybridisation is carried suspended during monsoon season and out effectively only during dry season for a period of six months from November to May. Some of the reasons attributed to the stoppage of pollination work during monsoon are the slippage of the trunk while climbing, problems in male flower collection and wetting of the pollination bags during heavy rains. Technological developments in coconut sector viz., the invention of coconut climbing devices and cryopreservation techniques have contributed to safe climbing and pollen storage for utilisation during off-seasons, respectively. However, the wetting of the pollination bag used for hybridization remains a problem. We report the development and successful initial testing of a durable raincoat for the pollination bag so that coconut pollination can be carried out throughout the year in heavy monsoon areas. This reusable raincoat is integrated with the ground pollination unit and can be fitted easily on the pollination bag, and it ensures complete dryness of the pollination bag. Only one climbing per tree is required for emasculation, bagging and raincoat fitting on a single inflorescence. The raincoat has provisions for suspending a pollen delivery tube and pollen dusting and bag removal can be done from the ground. The durable raincoat cover for the pollination bags has resulted in a fruit setting of 22.5-41.3 per cent.

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.