n vitro conservation of Plant Genetic Resources . It is an essential part of the overall strategy for the conservation and exchange of plant genetic resources worldwide. It is an alternative for the preservation of plant material for research purposes, for the massive propagation of plants and has allowed the promotion and development of in vitro genebanks that have contributed to the scientific community and the maintenance of important biodiversity.
Summary
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- 1 Story
- 2 Advances
- 3 Importance
- 4 In vitrogermplasm banks
- 5 Types of in vitrostorage
- 6 Short-term storage
- 1 Main factors
- 1.1 Temperature
- 1.2 Culture medium
- 1.3 Culture container
- 1.4 Modification of the gaseous environment
- 7 In vitrogermplasm banks in tropical crops
- 8 Source
- 1 Main factors
History
The conservation of plant germplasm as a scientific activity was proposed in the 70s of the 20th century with the aim of preventing genetic erosion and improving the agricultural productivity of many species by conserving different species and genes of interest. There are two basic strategies for plant germplasm conservation, in situ conservation and ex situ conservation .
In situ conservation, species are kept in their natural habitat, generally in national parks, biological reserves and ecological reserves. While in ex situ conservationspecies are preserved outside their natural habitat, in botanical seed banks, field plant banks or in vitro plant banks .
Botanical seed banks are very useful in sexually propagated species whose seeds remain viable for a long period of storage, but they should not be applied to conserve plant species with botanical seeds of short survival, nor can they be used in case of working with self-incompatible plants, or plants with forced vegetative propagation. In these cases, the genetic diversity of these species can be conserved by plant banks in the field or by in vitro conservation techniques .
Banks in field conditions have as a limitation that large tracts of land are needed, the costs for maintenance associated with agro-technical work are high, it is necessary to control pests and diseases, and they are also vulnerable to climatic disasters. For this reason, the development of new conservation techniques has made it possible to better preserve important genetic resources for many countries.
Advances
In particular, the advances achieved in in vitro cultivation in different plant species gave the possibility of developing new alternatives for ex situ conservation , by allowing enough individuals to be available in relatively short periods of time and facilitating the handling of plant material as plants. with a homogeneous physiological development. Since then, in vitro conservation of plant germplasm has been a working tool that supported the conservation of botanical seeds and conservation in the field, since it allowed us to have safe duplicates of those genotypes of particular interest.
This conservation route can be carried out through two methods: minimal growth and cryopreservation.
The method based on minimum growth is the most general, it is based on modifying the optimal growing conditions, to decrease the normal growth rate of the species under study, thereby reducing the frequency of transfer of the plants to the medium. of fresh culture.
Importance
In vitro conservation of plant germplasm offers the possibility of storing a large number and variety of samples in a small area and facilitates access to them for evaluation. Its aseptic conditions guarantee greater sanitation of the samples and consequently allow an increase in the exchange of healthy plant materials.
The main objective of in vitro germplasm banks is to conserve species that have short and low viability botanical seeds, vegetatively or clonally propagated crops, or that are highly heterozygous and require vegetative propagation to preserve their genetic integrity. Short-lived roots and tubers have also been included in the storage process, such as Solanum tuberosumL. (potato), Ipomoea batata L. (sweet potato) and Manihot esculenta Crantz (cassava).
In vitro germplasm banks
They are sites for the conservation of genetic resources under controlled laboratory conditions and that involve various cultivation and in vitro storage techniques . They seek to maximize the diversity of specimens collected from populations in the field or in their center of origin. The collection unit kept under controlled conditions can be botanical seed or vegetative explants, depending mainly on the growth habit of the species.
To provide explants and seeds with adequate in vitro storage conditionsTechniques have been developed that allow a high diversity to be maintained in small spaces, in aseptic conditions and safe from the environmental risks that could cause its loss. For example, Arachis retusa embryos and cotyledons were conserved in vitro to overcome the problems of low viability in the storage of sexual seed, due to the high lipid content.
Furthermore, in vitro culture facilitates the exchange of genetic material, since small samples can be sent under aseptic conditions, even to countries with very strict phytosanitary regulations.
Types of in vitro storage
In vitro storage can be classified, according to its duration, in “short-term storage” (known in English as “short-term storage”) whose objective is to reduce the growth rate of plant material, so techniques are generally used that promote reduced growth, and “long-term storage”, where cryopreservation is used to guarantee in vitro conservation for prolonged periods of time.
The minimal growth method has been widely used in practice for the conservation of germplasm, there are several examples of its application in the creation of in vitro banks . Among others,sp. (sugar cane), yucca and potato. This method is one of the main variants that can guarantee the success of a conservation program, because it is relatively simple and can be easily established with the equipment that normally exists in a tissue culture laboratory.
This method is also known as reduced growth because, as explained, it is based on the decrease in cell division and metabolism of the plant. Its objectives are to increase the in vitro longevity of the cultures without genetic changes taking place, therefore, there is not a total arrest of the cellular processes but a decrease in the speed with which they occur and thus the frequency of transfer of the plants in fresh culture medium.
Modification in the composition of the culture media is one way of limiting growth, for example, the in vitro growth of cassava plants decreased proportionally with the total nitrogen content of the culture medium; concentrations below 10mM, in terms of number of stems and knots per stems were harmful. In vitro sugarcane
plants have been preserved, with quality and for six months without subculturing, with the reduction of the salts proposed by Murashige and Skoog (1962) between 25 and 50% of their normal concentration.
Another option may be to increase the osmotic concentration of the culture medium, the limitation of growth due to this effect is due to the reduction of adsorption of water and nutrients from the culture medium. Sucrose, as it is highly metabolizable, can act as an osmotic agent in high concentrations. Other non-metabolizable osmotic agents, such as mannitol and sorbitol, are more effective in limiting growth, because they interact with sucrose content and storage temperature.
The intensity and quality of light are other important factors in the control of the growth rate, especially in its interaction with temperature, which, as has already been expressed, is another of the factors most used in conservation by the method of minimal growth.
Short term storage
In short-term storage, the explants remain in vitro for up to 12 months, managing culture conditions to delay growth and increase the intervals between subcultures.
As part of the strategies used to decrease the growth of the explants and increase the intervals between subcultures, there is reducing the temperature of the growth rooms, modifying the culture media and other environmental factors that must be taken into account to optimize storage. .
Main factors
Temperature
By lowering the cultivation temperature, there is a reduction in metabolic activity and, consequently, in the growth of the explants (this temperature depends on the requirements of each species). Temperatures around 4 ° C are generally used for temperate climate crops and between 10 and 15 ° C for tropical germplasm. Temperature control is usually combined with other factors to achieve reduced growth. In banana, reduced temperatures have been combined with reduced light intensity or complete light suppression. Vegetative propagation of banana stalks is normally carried out at 22 ° C with a light intensity of 3000 lux. However, for short-term storage, they are placed at 15 ° C under a light intensity of 1000 lux.
If the explants are kept at low temperatures (below 4 ° C) for prolonged periods, physiological damage can occur, caused by the cold, which affects changes in metabolism, protein content, and the composition and function of the membranes. These problems can be reversible when there is no prolonged exposure to this condition. Tropical plants are generally sensitive to cold damage and the storage temperature depends on the particular sensitivity of each species. For example, the optimal storage temperature for encapsulated microtoss of pineapple ( Ananas comosusL.) is 8ºC for a period of 45 to 60 days. In non-tropical species, the reduction in temperature often acts as a signal to break the resting state of the explants and induce the activation of their growth. For example, the storage of lily ( Lilium longiflorum L.) at low temperatures (0–10ºC) induces the break of the resting state, which consequently leads to its germination and flowering. Therefore, its in vitro storageit must be carried out at 25ºC, in a culture medium with all the mineral salts at a quarter of its concentration and vitamins from the Murashige and Skoog (1962) (MS) medium and a high concentration of sucrose (9%) for 28 months. In addition, temperature management can be combined with a reduction of the nutrients available in the culture medium, an aspect that is also of great importance. Both measures slow down the growth process and prolong storage.
Culture medium
Reducing the concentration of metabolizable mineral elements and / or carbohydrates (eg, sucrose) in the culture medium may be an important strategy for reducing explant growth. Other measures may be increasing the osmotic potential of the medium (especially through the use of non-metabolizable carbohydrates, such as mannitol), the use of higher concentrations of gelling agents, the addition of certain growth regulators (such as abscisic acid [ABA]) , or other substances (such as magnesium chloride) to reduce damage to the explant. As a consequence of the above measures, the explant absorbs nutrients more slowly and a reduction in growth occurs.
Culture container
The volume of the culture container can be decisive in defining the frequency of subcultures and the optimal storage of explants.
Modification of the gaseous environment
Growth reduction can also be achieved by decreasing the level of oxygen available for explants. However, under these storage conditions, hyperhydric, necrotic, and slower growing tissues often develop. The hyperhydricity is a physiological disorder which is characterized by the glassy appearance and swollen tissue, turgid, aqueous and hipolignificados stems and translucent, green, brittle and deformations in the cuticle and epidermis organs. Explant necrosis, due to reduced oxygen, has been reduced by adding silver nitrate, gibberellins, fructose, calcium, ascorbic acid and / or activated carbon to the culture medium.
One method of modifying the gaseous environment is to lower the oxygen partial pressure using controlled atmospheres or to lower the atmospheric pressure in the chamber. This has allowed in vitro storage of tropical species sensitive to low temperatures. Another method to limit the level of available oxygen is to use a layer of mineral oil or liquid medium to cover the explants.
In vitro germplasm banks in tropical crops
Most in vitro germplasm banks specialized in tropical plants are associated with international research or conservation centers and, to a lesser degree, with universities. An example is the Tropical Agricultural Research and Teaching Center (CATIE), in Costa Rica, where work has been done to develop appropriate protocols for the in vitro storage of zygotic embryos, somatic embryos, apices and seeds of different coffee genotypes, as well as cell suspensions of this same species and of Musa spp. This, in addition, has facilitated the exchange of plant genetic resources with producers and with international organizations.
CIAT has preserved corn ( Zea mays ) and Cassava (Manihot esculenta ); the International Potato Center (CIP) local potato varieties, the IITA yam bean ( Sphenostylis stenocarpa ), cowpea ( Vigna unguiculata ), bambara peanut ( Vigna subterranea ), soybean ( Glicine max ) and cassava ( Manihot esculenta). Also, member institutes of The Consultative Group on International Agricultural Research (CGIAR), which collaborate with official organizations in different areas of the Middle East, America, Asia and Africa, are mainly dedicated to scientific research. to promote sustainable agricultural growth that strengthens food security and, as a fundamental task, they have to maintain international genebanks that preserve and facilitate access to genetic resources.
The Biodiversity International group (previously known as “The International Board for Plant Genetic Resources” [IBPGR]), with its different headquarters in America, Asia, the Pacific, Oceania, Europe and Africa, has focused on the in vitro conservation , through slow growth, of cacao stems, mango, banana, plantain, avocado, potato (from CIP through material exchange), cassava, Allium species , minitubers of yams ( Dioscorea spp.) ( from IITA), tiquisque ( Xanthosoma spp.) and malanga, taro, chamol or ñampí ( C. esculenta ) and some forage species ( Cynodon spp. and Digitariaspp.). In addition, BI has used cryopreservation to store zygotic coconut embryos, cocoa beans, avocado seeds, sweet potato and potato meristems, sugar cane explants ( Saccharum officinarum ) and Citrus spp. Regarding staple crops for food in Africa and Asia, work has been carried out mainly with Allium spp., Corn and rice. The International Network for the Improvement of Banana and Plantain ([INIBAP / IPGRI] by its English name “International Network for the Improvement of Banana and Plantain, Belgium) specializes in maintaining an in vitro germplasm bankbanana and has made efforts to eliminate diseases during storage. For example, by cryopreservation, cucumber mosaic virus was removed in apical meristems of this species. The universities of Taiwan and Cambodia established an in vitro citrus germplasm bank , in conjunction with the Long Dinh Center for Fruit Research (LDFRC) in Vietnam. New varieties with tolerance and resistance to various diseases were generated from plant genetic resources conserved in this bank. The bank has 15 varieties of orange, 11 varieties and five wild lemon species, 45 grapefruit varieties and more than 27 varieties belonging to six species and hybrids.
In Asia, the Food and Fertilizer Technology Center (FFTC) uses a genebank to save wild rice and banana materials from extinction so they can be used in genetic improvement programs.
There are other smaller and more limited efforts, which also focus on tropical crops and that seek food security and the development of methodologies for poorly investigated species, such as cryopreservation of several species of the genus Dioscorea (with potential as a substitute for potatoes) in the Institute of Plant Genetics and Plant Crop Research, Gatersleben, Germany, apical sweet potato meristems at the Kagoshima Institute of Biotechnology, Japan, and sugarcane stems from somatic embryos by slow in vitro growthwith subcultures every two months in the germplasm conservation laboratory of the School of Biology and Conservation Sciences of the University of KwaZulu – Natal, in South Africa. In Cuba, the Research Institute of Tropical Vias ( INIVIT ) is in charge of the conservation of germplasm of tropical roots and tubers, plantains and bananas) and maintains, in addition to field collections, in vitro collections of these plant species. With the use of mannitol they reduce the growth of the in vitro plants of cassava , yam , taro ( Xanthosoma spp.) And sweet potato . In general, gene banks in vitro they are a useful tool for the conservation of genetic resources of tropical crops of agricultural and nutritional importance.
