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International Journal of Life Sciences N. Manvelidze et. al., Vol. 5 No. 4 ISSN: 2277-193x
International Journal of Life Sciences. Vol. 5 No. 4. 2016. Pp. 190-193
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Full Length Research Paper
The Induction of Callusogenesis and Morphogenesis of Staphylea colchicaintoin vitro culture
N. Manvelidze1, N. Zarnadze2, N. Varshanidze2, I. Diasamidze2, S. Manjgaladze3, T. S. Bolkvadze3 and K. Dolidze2
1Master student, Batumi Shota Rustaveli State University, Georgia.
2 Professor,Batumi Shota Rustaveli StateUniversity, Georgia.
3 Researcher, Batumi Shota Rustaveli StateUniversity, Georgia.
Abstract
Introduction
In experimental botanics and phisiology the opportunity of hormonal regulation of plant growth and organ formation by methodic operations of tissues and separate cell cultivation is considered to be the greatest phenomenon. With these methods proceeds mass regeneration of plants in vitro conditions, which has plant cell totipotency as a basis. Atotipotency in vitro conditions provides the production of plant-regenerants identical to mother plant (1;2), or getting calluse cultures and indusing new regenration variants from it on the basis of dediferention proceeding in culture, which represent a starting material for the selection. (4) The object of our study was one of the relict and endemic species -Staphylea colchica.
Staphylea colchica is a representative of Staphylea type family. It is a bush or a small tree (up to 5 m height). It is a vulnerable plant from the red listand thus, because of biotechnological method advantages, studyingits reproduction peculiarities using nontraditional ways is expedient in order to create sapling material (3).
Material and Methods:
The object of the study was Staphylea colchica spread in Batumi Botanical Gardens. For the in vitro cultivation we have isolatedvegetation sprouts, and for callus genesis we have used 5-6mm fragments of leaves taken from sterilized cultures. The sterilization of primary material proceeded with 0.2% diocide water solution for 20 minutes of exposition.Explant cultivation proceded on different modification nutrition areas of Gamborg (B5), which were different mainly according to hormone nature, concentration and their correlation. Nutrition area pH was 5,8-5,9,for callus induction we have used auxines: indolebutyric acid, naphthoylacetic acid, and causing organogenesis proceeded by using cytokines – benzylaminopurine and 2-izopentiladinine, independently and in correlation with auxines as well.
For callus induction, cultures incubated in thermostat, darkness, 26-270C temperature, and for organogenesis realization light was required. 2-3 Klux of light, with 8/16 hour phytoperiod and 26-270C temperature. We registered the results every 25-30 days, we determined the frequency of callusogenesis and regeneration in percents, the growth intensity of callus by 5-point system, in each variant we examined 20 explants two times.
Result and Discussion
In order to study callus regeneration potential, we moved part of calluses on organogenic nutritious areas, which had same mineral consistency and different hormonal balance, namely, cytokine totally exceeded auxine, in some cases auxine was totally excluded from the consistency of the nutritious area.
For regeneration inductionwe put cultural vessels with explants in the light of 16/8 hours of photoperiod at 2710C temperature. On organogenic nutritious areas Staphylea colchicacalluses had changes ofvarious types,namely:
1)From callus tissues reproduced on nutritious areas containing NAA:
a)Some of them continued growth and development, grew in size, became light green and induced one or two regenerants;
b)The most part of callus tissues, about 70-80% gave mass regeneration.
2)From callus tissues reproduced on nutritious areas containing IBA:
a)Some continued proliferation;
b)Tissues induced roots;
c)75-94% gave mass regeneration;
Morphogenetic potential of those callus tissues, which did not undergo any organ-producing process and grew intensely, was defined only by formation of histological structures.
Mass regeneration of buds from callus tissue, on the one hand, depended on the factor of which auxine consistency area produced the primary callus, and on the other hand, which cytokine and what extent of the concentration was in the consistency of organogenic nutritious area. As the study results have shown, calluses induced by the influence of IBA were characterized by the highest morphogenetic potential. And those calluses that were produced on nutritious area containing NAA got behind this indicator.
Strengthened induction of stalk morphogenesis was noted on the nutritious areas with 2-IP and IBA, (photo 2) where the correlation was 12:3mm; 15:3mm accordingly. Mentioned concentrates represented optimal amount of hormones. Regeneration frequency was 75-94%, the general number of regenerated buds on 20-25 diameter callus tissue - 57.5%, concentration increase of 2-IP (18-20mm) strengthened regeneration on callus, i.e. development of already generated organostructures and buds. Regeneration frequency was 82-100%. And the total amount of buds was 59,7-164,0. By the influence of 2-IP, on the callus tissues there formed new morphogenetic knots.
The formation of new places of calluses proceeded in heaps, from which simultaneously developed 6-7 apical parts of germinal buds and afterwards buds. Bud formation on callus proceeded asymmetrically and asynchronously. They gradually overtook each other in growth and development. The distribution of regeneration bud groups on new and same consistency nutritious area lead to the fastened development of sprouts and buds having normal phenotype. 2-IP increase up to 22-25mm concentration more caused the formation of large number of morphogenic knots than organogenesis on callus. It was necessary to move formed morphogenic knots on nutritious area consisting low concentration hormones (5mm), in order to grow buds in height.
Similar answer of explants was noted on the nutritious area both with 2-IP and NAA. We must note that regenerated buds on nutritious area containing 2-IP+IBA, rooted better than buds on nutritious area containing 2-IP+NAA.
We must also note that in order to show morphogenetic potential, the usage of BAP instead of 2-IP appeared to be effective as well. On the callus, the development of morphogenetic knots and buds proceeded quite intensively. Regeneration induction was noted from 10-12th day of explant sowing, but from the 20-th day the explant was totally covered with buds. The usage of nutritious areas containing IBA and BAP correlation showed that the correlation of their concentration have influence on regeneration coefficient.
Fig. 1 NAA and BAP different concentration influence on callus growth and development
Fig. 2 NAA and BAP different concentration influence on calluse formation frequency
Photo 1.Callus development on nutritious area B5+BAP 2mm+IBA 15mm Photo 2. The development of morphogenetic knots and buds
On nutritious areaB5+2-IP15mm+NAA 3mm
For plant reproduction into in vitro conditions it is necessary to define the optimum of those physical conditions which will provide effective result. Physical conditions of the cultivation are: the regime of temperature, darkness, light intensity and duration. Culture incubation for organogenesis proceeded according to the following regime:
a)With phytoperiod – 16 - hour light : 8-hour darkness;
b)24-hour non-stop light.
Explant cultivation in experimental conditions showed that it is better to cultivate in the first regime conditions, namely with the photoperiod of 16/8 hours. Regenerated explants had sharp green coloring, beautiful habitus and lancet like leaves.The intensive bud formation, activating meristemplaces, proceeded in non-stop light conditions as well, though it is worth to note that, this time, the development of apical sprouts compared to the phytoperiod, was slow. The formed sprouts had green coloring, thickened stalks and shortened internodes.The optimal temperature for explant incubation, as well as for many other cultures, was 26±10C. Temperature interval of phytotrone varies between 25-270C. On higher or lower temperature than this interval, plants showed contraindication of growth and development processes. They did not grow or the growth intensity was minimal. Thus, the experimental study showed that in vitro method of isolated tissue, cell and organ can be successfully used in mass regeneration.
Conclusion
According to the conducted study we have determined that for callus induction and further growth and development, it is essential to add auxine phytohormones to the nutritious area, as on hormoneless areas explants not only did not callus but badly increased. At the end of sub-cultivationthey became rough and black, or ten days later they degenerated and died. The frequency of callus creation and proliferation intensity depended on the nature and concentration of auxines. During cultivating leaf explants, from the 8-12-th day of sowing on most analyzing nutritious areas, callus tissue was induced. We must note that callus formed by the influence of different auxines was characterized with various morphology and growth intensity. On nutritious area, containing naphthoylacetic acid, hard and whitish tissue developed, and by the influence of indolebutyric acid, there developed white dedifferentiated tissues of average hardness (Photo 1).
The analysis of growth intensity has shown that callus induced on any studied concentration of indolebutyric acid and naphthoylacetic acid (5; 8; 12; 15; 20 mkm) but the intense proliferation was noted on nutritious area consisting naphthoylacetic acid (4-5 points). (Diagram 1) The best one appeared to be the nutritious are with 15-20mmconcentration. Callus formation frequency was 82,6-93.2%. At the end of the second sub-cultivation there formed callus tissue of large classical morphology, which consisted of well-dedifferentiated cells. (Photo 2)
The intensity of callus growth increased with the increase of auxine concentration. Despite the fact that at the beginning (0- sub-cultivation) callus initiation was to a certain extent slow, further growth and development preceded simultaneously and during all further sub-cultivation it proceeded more intensively. Accordingly, callus biomass accumulation was in direct proportion with the number of re-sowing.
Callus forming induction grew with the introduction of cytokines with auxines into the nutritious area. Cytokines and auxines participate in cell growth, division, stabilizing organogenic features of callus, in the process of sprout differentiation and bud formation.
References
Alasania N.Sh;Zarnadze N; Lomtatidze N; Nakashidze NMorphogenetic Features of Passiflora in vitro sistem //Georgia, Subtropical crops, . 2010. Vol. 1-4; pp. 127-133.
Egorova N. A. The Role of some factors in the process of callusogenesis induction into in vitro of etherbearing plants / N. A. Egorova, I. V. Stavtseva, O. V. Iakimova (and others) // Factors of experimental evolution of plants: Зб. sc. paper. – Kiev: УТГіС, 2014. – V. 15. – pp. 63–67.
Manvelidze Z. Ecosystem diversity of Ajara Forest. Biodiversity of Ajara (South Colchis), Publishinghouse 2009. BSU, Batumi.Pp 550-564.
Zarndaze N. Zh.; Jakeli E.M.; Chelidze N.; Gamrekelidze Kh. O.; Clonal micro reproduction of plants Vaccinium uliginosum L into in vitro // Introduction, preservation and monitoring of plant diversity / Materials of international conference “for the 175th anniversary of the Botanical Garden named after Academic A. V. Fomin of Kiev Taras Shevchenko National University”/ 2014, 20-24 May, Kiev.
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