- •2) Objects and methods of animal biotechnology
- •3) Totipotent, multipotent, pluripotent animal cells
- •5)The principles of genetic cloning
- •6.Allophenic animals. Genetic chimers
- •8) Methods for introducing foreign dna into animal cells
- •9)Cryopreservation of reproductive and germ cells of animals and humans
- •11)The principles and methods of plant cells cultivation in vitro
- •14)Differentiation and dedifferentiation in plant cell culture. The obtaining callus mass and cultivation of callus tissue .
- •15)The influence of phytohormons on morphogenesis and regeneration in plant cells culture
- •16.The main path of morphogenesis processes in plant cells culture
- •20) The factors influenced on microclonal propagation in plant cell culture.
- •21) What is Biotechnology? Various definitions of “Biotechnology”. History of Biotechnology
- •22.Microbial Biotechnology: fundamentals of applied microbiology
- •26) Somaclonal and gametoclonal variation in plant cells culture.
- •27) Artificial seeds". Embryo culture in vitro
- •28. Culture of apical meristem cells
- •29)Cell reconstruction. Theoretical means of cell reconstruction
- •32) Main objects of animal biotechnology:
- •33) Morphological and functional features of gametes - eggs and sperm
- •39) Basic approaches and principles of gene therapy. Gene therapy ex vivo, in vivo, in situ.
- •35)The history of investigations of the genetic transformation of animal cells
- •53)Genetic engineering. Methods of genetic transformation
- •56) The vector systems used in the genetic engineering
- •62) Methods of producing chimeras
- •57) Methods of genetic engineering: agrobacterial genetic transformation
- •63) Collection and cultivation of oocytes in vivo and in vitro
- •68) Draw a diagram of the structure of plasmid pBr322
- •69) Draw a diagram of an experiment in genetic engineering (design recDna) and give a description of the main stages
- •74) Modes of freezing and thawing of gametes and embryos
- •75) Methods of artificial fertilization: gamete insemination fallopian tube (gift), zygosity insemination fallopian tubes (zift).
- •80) Methods of animal cloning, reproductive and therapeutic cloning
- •81) Microorganisms in water and wastewater treatment
- •86) Use of e.Coli for the biotechnological production
- •87) Microbes in milk and dairy products
28. Culture of apical meristem cells
Meristem - cone actively dividing cells located at the tip of roots or shoots. For the recovery of used meristem shoots width of 0.1 mm and a length of 0.25-0.3 mm. The method is based on the fact that the distribution of plant virus and uneven concentration is lowest in the region of the apical meristem. The isolation of the apical meristem and drop it into the culture medium results in a reduction or complete elimination of the concentration of the virus in the child after its plant regeneration from the apical meristem.
There are several theories to explain the absence of virus in the meristem: the slow distribution of viruses in actively dividing cells, inhibition of virus multiplication high concentration of auxin, the impact of the components of medium.
The authors considered the method of J. Morel and S.Martin (1952), first received free of virus from an infected plant dahlias donor plant. The current method is widely used and is used for the recovery of seed potatoes, fruit, berries, ornamental plants. The use of the apical meristem in itself does not guarantee the compulsory recovery. The virus can be present in the apical meristem in a latent state. In connection with this method must be combined with other apical meristems recovery methods (thermo-and chemotherapy) and used to control viral infection of a plant regenerated electron microscopy and / or immunoassay. There have been instances when free of viruses were obtained from the regenerated carrier viruses apical meristems. It appears that the loss of virus in this case connected to a combination of factors arising from the cultivation of meristems (composition of the nutrient medium, the presence of hormones, etc.)
What factors contribute to the elimination of virus in the culture of meristems? One major factor is the size of the meristem. It is known that the efficiency improvement is inversely proportional to the size of the meristem, and the efficiency of plant regeneration from the meristem is directly proportional. Therefore, for each plant selected the optimal size of the meristem, which provides a high level of elimination of viruses and acceptable way of regenerated plants.
Apical buds provide higher efficiency in comparison with the regeneration side (effect of apical dominance). Influence of season and physiological state of the plant on the regeneration efficiency was seen earlier. For example, the optimal period for the potato tuber after the quiescent state. Explants (shoot tip) used for the isolation of meristems sterilized in 75-95% ethanol and / or 0.1 - .0.5% sodium hypochlorite, followed by repeated washing with sterile water. Disentangling the meristem is produced in a laminar hood under sterile conditions under a binocular microscope. The articulation of the meristem size 0.5 - + 0.2 mm.
For the cultivation of apical meristems are usually used modified Murashige and Skoog medium, picking up the hormonal composition is optimal for culture. Applies a low concentration of growth regulators (0.1-0.5 mg / l); auxins may be necessary for the formation of roots, cytokinins and auxins - to stimulate cell division, gibberellic acid is sometimes added to lengthen the shoots. Normal meristem culture is the temperature of 21-25 0 C for bulbous plants requires a lower temperature. The optimal length of the day - 14-16 hours.