Scientific Journal of KubSAU

Effects of different levels of vitamins (thiamine, pyridoxine and nicotinic acid) and macro-elements (MS, NN and PG) on development of globular, heart- and torpedo-stage embryos and plantlets with green cotyledons and on shoot production from them were studied in grapevine interspecific hybrids: cvs. ‘Bianca’, ‘Podarok Magaracha’ and ‘Intervitis Magaracha’. Pro-embryogenic calli were derived from petiole explants on solid full-strength NN medium supplemented with different levels of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA) for each cultivar and used to initiate cell suspensions in liquid NN medium supplemented with 1 mg L-1 2,4-D and 0.2 mg L-1 BA. The following formulations of media were optimal for different stages of somatic embryogenesis and for plantlets with green cotyledons development: liquid NN medium supplemented with 0.5 mg L-1 BA  for globular embryo formation; liquid HTE medium (with PG macro-elements, thiamine and pyridoxine at 5 mg L-1 each and 0.5 mg L-1 nicotinic acid): supplemented with 0.2 mg L-1 BA  for heart-stage embryo development, supplemented with 0.1 mg L-1 indole-3-acetic acid (IAA) and 30 mg L-1 sodium humate  for torpedo-stage embryo development and supplemented with 0.5 mg L-1 gibberellic acid (GA3)  for the growth of plantlets with green cotyledons. Solid MS medium modified by the addition of nicotinic acid, pyridoxine and p-aminobenzoic acid (PAB) at 5 mg L-1 each, 0.5 mg L-1 thiamine and 0.5 mg L-1 BA was best efficient for shoot production from plantlets. Inflorescence formation in regenerated plants of cv. ‘Podarok Magaracha’ grown on solid hormone-free PG medium was induced by addition of nicotinic acid, pyridoxine and PAB at 5 mg L-1 each, 0.5 mg L-1 thiamine and 0.2 mg L-1 BA into liquid modified HTE medium for growth of plantlets with green cotyledons and into solid modified MS medium for shoot production from them.

Nutrient media for shoot development from shoot tips 0.5-0.8 mm with several leaf primordia in four grapevine genotypes were optimized by means of a mathematical design of experiment. Over the range of component concentrations CaCl2 had considerable the highest effect on shoot tip development compared to other components of the media. Grapevine genotypes were different in their needs for concentrations of macro-elements to optimize the process of their growth and development. Shoot development from shoot tips in each grapevine genotype as a function of macro-element concentrations in media must be described by an individual regression equation. The proposed method of result evaluation and the mathematical design of experiment may be used in physiological and agricultural research for optimization of processes affected by numerous factors. The optimized media for shoot tip development may by used for sanitation of grape plants from viruses in meristem cultures

Two methods were developed, one to assess experimental results by reducing a number of developmental parameters to the overall quality, leading to the overall quality criterion, and the other to optimize processes affected by numerous interacting factors, in-vitro plant development in this case, by applying a mathematical design of experiment. Single-bud cuttings with one leaf of two Vitis vinifera L. genotypes were excised from the central part of two-month-old in-vitro grown plants and used as explants. The explants were established on bridges of filtering paper in liquid media and on solid media. Eighteen modifications of these media contained five macro-elements, each at three concentrations, and the distribution of these macro-element concentrations followed the law of random numbers (Experiment I). Parameters characterizing arm, leaf and root development of two-month-old plants of each study genotype established on each liquid or solid medium were reduced to the overall quality of plant development. Since the study genotypes differed in the ability to grow on liquid and solid media with different macro-element concentrations and also in the ability to utilize these macro-elements, the dependence of in-vitro plant development on macro-element concentrations was described by different regression equations. That is why the regression equation describing the average outcome of plant development in the two genotypes on liquid and solid media was not as significant and the description of the process was not as adequate (determined) as regression equations calculated for each process. The regression equation which describes the dependence of the average outcome of plant development on macro-element concentrations in Experiment I is as follows: y5 = 0.027 + 0.116x22 + 0.109x2x4 + 0.106x2x3 + 0.114x4. Stepwise calculation of macro-element concentrations to optimize in-vitro plant development was done based on macro-element concentrations of the initial medium of Experiment I as starting points since that medium was best efficient for this purpose by using regression equation (y5) and algorithms of multiple curvilinear stepwise regression according to the Box-Wilson method of steepest ascent. Experiment II was undertaken where macro-element concentrations (‘steps’) were calculated in a stepwise manner to optimize in-vitro plant development of the two V. vinifera genotypes and the rootstock ‘Kober 5BB’. This led to a liquid medium and a solid one which enabled a better plant development in the three genotypes relative to the use of controls: media with ½ MS macro-elements and the initial medium whose macro-element concentrations entered as starting points to calculate ‘steps’ for optimization of in-vitro plant development. The optimized medium contained macro-elements: 318 mg l-1 NH4NO3 (x1), 1188 mg l-1 KNO3 (x2), 370 mg l-1 MgSO4 7H20 (x3) (MS), 370 mg l-1 KH2PO4 (x4), 331 mg l-1 CaCl2 (x5) (MS), and other substances at optimum concentrations adjusted earlier: ¼ MS Fe-EDTA, ¼ MS micro-elements, 20 mg l-1 myo-inositol, 0.1 mg l-1 thiamine (MS), 0.5 mg l-1 nicotinic acid (MS), 0.2 mg l-1 pyridoxine, 2 mg l-1 glycine (MS), 0.1 mg l-1 indole-3-acetic acid, 10 g l-1 sucrose and, only for solid media, 7 g l-1 Difco agar. The optimized medium may be used for propagation of virus-free plants, valuable clones and grapevine genotypes created by gene engineering. The mathematical design of experiment reported in this paper which enables stepwise optimization of in-vitro plant development may be used both in agriculture and in the food industry