EXOGENOUS REGULATION OF TUBERIZATION OF Solanum tuberosum L. IN CULTURE in vitro (review)
A.N. Deryabin, N.O. Yur’eva
The authors discussed the effect of temperature, photoperiod, composition of carbohydrates, content of nitrogen in nutrition medium and also growth regulators (auxin, cytokinins, gibberellins, abscisic acid, ethylene, retardants) on tuberization in potato plants cultivated in vitro.
Key words: Solanum tuberosum L., microtubers, stolon development, tuberization, sugars, photoperiod, temperature, phytohormones, retardants.
Tuberization (TB) in potato (Solanum tuberosum L.) is a highly coordinated process, involving morphological, physiological and biochemical changes in plants at different ontogeny stages. The following TB stages are allocated: induction and initiation of a stolon; stolon growth and branching, stolon growth cessation; induction and initiation of a tuber, tuber growth and maturation (1-3).
According to M.Kh. Chaylakhyan (4), TB includes two phases: 1st - the formation and growth of stolons, 2nd - the formation and growth of tubers. Even in 1964, R.G. Butenko has reported that numerous factors, their additive or multidirectional impact indicate the complex interaction between plant organs during the TB start of potato in vitro (5). The inverse correlation between a stem growth and TB has been revealed, which is typical for both plants grown in vivo and for culture in vitro: stem growth slows down and stops with the start of TB (6). The dynamics of TB phases in different potato varieties cultivated in vitro can be described as a sigmoid curve, which performs periodic decreases in stolon growth rate owing to formation of new metameres (internodes); the formation of tubers is discrete, it takes 3-4 weeks with the consequent raise of tubers weight (7). TB can be observed in potato plants grown in vitro even in stress conditions: at the depletion of nutrient medium, the alternation of contrasting light conditions, under shock effects of physical factors (8) including cultivation of plants regenerants in the light (9).
Exogenous non-hormonal factors affecting tuberization. Carbohydrates composition of nutrient medium. Even in 1953, W.G. Barker has showed that bringing of etiolated potato shoots into the culture medium with 8% sucrose induced TB (10). Later, the direct correlation between TB intensity in vitro and sucrose content in a medium has been revealed (11-13). According to F.F. Weering (14), the high content of sucrose - 8% vs. 4% - increased the number and weight of microtubers, however, the increase of concentration by 12% was not correlated with a number of new microtubers (15). According to other authors, the optimum for TB content of sucrose varies within 6 - 8%, and concentrations below 4% and above 10% cause TB disturbance or formation of small microtubers (16, 17). Apparently, sucrose at a concentration of 8% was considered as the most suitable carbohydrate for growing potato in vitro, and the osmotic substance necessary for obtaining microtubers as well (18, 19). It has been proved that disaccharides are necessary for the formation of microtubers, although plants are capable to absorb monosugars too. Studying the influence of carbohydrates qualitative composition on TB, it has been shown that 8% glucose provides the earliest start of TB compared with 8% sucrose and 8% fructose (20). V.N. Ovchinnikova (6) has revealed the necessity of introduction to the nutrient medium for TB of sucrose (4-6%) and fructose (2%). Some authors supposed that the high osmotic pressure in the nutrient medium with a mixture of carbohydrates (4% glucose and 4% fructose) promotes the formation of smaller microtubers than in the medium containing only 8% sucrose (21). Consequently, varying the qualitative and quantitative composition of carbohydrates in the medium makes possible to regulate individual stages of TB (20, 22). It is known that autoclaving leads to some changes in the initial qualitative carbohydrates composition due to a partial decomposition of sucrose to glucose and fructose (23), which increases osmotic pressure in the solution. However, no differences were found in the number of microtubers formed after adding of 8% sucrose to the medium before or after autoclaving (24). These data suggest that under in vitro conditions, sucrose is not only a source of energy necessary for growth of plants and microtubers, but it is also the optimal osmotic component of culture medium (25).
Nitrogen content. Have analyzed the few literature data, this can be concluded that the Murashige-Skoog medium (MS-medium) has the optimum nitrogen content for TB of potato in vitro. The reducing of total nitrogen content in MS-medium compared with a standard level resulted in the increased TB rate (26), but the less number and weight of microtubers (15), while the excessive total nitrogen in the medium disrupted TB (26).
Temperature. There were quite numerous studies of temperature effects on TB of potato in vitro, but no single point of view about the temperature optimum for the process: the optimum was considered 20-25 °C (27-29), 14-29 °C (30), not higher than 15 °C (31, 32) or 18-20 °C (19).
The effects of critical temperatures have been studied in a few works only. In early TB phase, high temperature levels inhibit growth of tubers and cause them to “stem-growing” (secondary growth) (32-34). It was experimentally proved that TB completely breaks at 4 °С (35) and 35 °С (16, 27). The greatest number of tubers were formed at 17 C°, but the higher temperature (22 oC) inhibits TB (36) and the low one (10-15 °C) stimulates formation of stolons in test-tube plants (31); though, stolons branching was observed only at higher temperatures (37). Our study has shown that exposing the initial potato stem explants to low positive temperatures synchronizes cell division in intercalary meristem and leads to formation of tubers leveled by their physiological age (38, 39).
Photoperiod. The first work on TB induction of potato in vitro was published more than fifty years ago (10), and it describes the possibility of tubers formation in conditions of continuous darkness and in the light as well. Later, R.G. Butenko (5) has shown that in vitro culture exposed to the short 9-hour day performs the higher TB rates compared with the long 16-hour day. It has been proved in different potato varieties that a short 8-hour day stimulates TB (19, 32, 40, and 41), inhibits stem growth and rhizogenezis (32).
Photoperiod primarily affects via the change in contents of endogenous phytohormones (42), which allows using photoperiod regulation of TB phases. The effects of daylight duration are unequal for potato varieties of different ripeness groups (26, 43). Early ripening varieties can form tubers in long days, while the short day is preferable for medium-late ripening ones (15). Bringing test-tube plants into the dark after growing in the 8-hour daylight stimulated the active TB regardless of ripeness group (44). Other researchers (45) recommend accelerating TB by alternation of artificial and natural lighting. The initial cultivation of stem explants in the dark provided formation of stolons without passing the phase of test-tube plants, which further makes possible to induce TB directly on the stolons (46).
Hormonal regulation of tuberization. TB of potato involves all the known phytohormones (auxins, gibberellins, abscisic acid, cytokines and ethylene) (42), which operate in dynamic equilibrium highly sensitive to chemical and physical factors. The effect of synthetic growth regulators is based upon imitation of phytohormones activity, or they influence plants’ hormonal balance (47). Studying the role of phytohormones in potato stem cuttings, it was found that the cuttings’ ability to form tubers depends on their location in a stem (tier) (48): the cuttings from an upper part of a stem did’t form microtubers; the lower tier was cut, the higher ability to form microtubers it performed. In authors’ opinion, the revealed dependence of TB from cuttings location in a stem can be explained by phytohormones gradient. The most active TB was observed in the cuttings taken from the middle and basal part of a stem (31, 49).
Auxins. The effect of plant growth regulators - auxins - on TB of potato has been shown in literature: indoleacetic acid (IAA) and 2,4-dichlorophenoxyacetic acid (2,4-D) are able to stimulate this process (14, 50), while the medium without 2,4-D provides formation of larger microtubers (51). The adding of IAA to a medium resulted in formation of microtubers of 1,5-3,0 times greater size than in the variant without the hormone (52), and adding of 2,4-D stimulated the increase in number of stolons (51). Varying concentrations of indolebutyric acid (IBA) and gibberellic acid (GA), it is possible to regulate stolon growth from axillary buds of stem explants (53).
Cytokines. R.G. Butenko (5) was the first who described stimulating effect of 6-furfurilaminopurine (kinetin) on TB of potato. In the short day, kinetin at a concentration of 2.0 mg/l inhibited stem growth and formation from axillary buds of stolons with microtubers. The presence of cytokines in nutrient medium changed the location of formation the microtubers (eg, potato stem explants grown in continuous darkness and affected by exogenous cytokines form tubers on stolons) (54). According to V.N. Ovchinnikova (6), 6-benzylaminopurine (6-BAP) and kinetin promote formation of tubers on stolons, and TB can be observed only in the middle or basal part of test-tube plants. Combined application of kinetin and jasmonic acid stimulated TB and simultaneously inhibited rhizogenezis (55).
Cytokines inhibit rhizogenezis in stem cuttings and suppress stem growth (6), thus increasing TB efficiency (56). The highest content of cytokines was detected in leaves of potato plants on the 4-6-th days after TB induction by a short day, and then cytokines content decreased in leaves against a background of its raise in stolon apexes (42). High tissue level of cytokines is associated with the intensive growth of an organ, as these hormones have an attracting effect (57). Some authors applied the cytokine 6-BAP at concentrations from 2 mg/l (58, 59) to 10 mg/l (60, 61) to study TB of potato in vitro. 6-BAP at a concentration of 2 mg/l and above inhibited rhizogenezis and stimulated the earlier TB than in control (without 6-BAP) (59). The highest number of microtubers was obtained applying 6% sucrose and 6-BAP together (62). Kinetin (63) and 6-BAP stimulate TB by increasing cell division rate, while a low temperature inhibits this effect (27). Cytokines accelerate the formation and growth of microtubers, stimulate thickening of stolon apex, but they do not affect TB process itself (64), therefore, they can‘t be considered the TB-inducers (28).
Gibberellins. The effect of exogenous gibberellic acid on TB of potato plants in vitro is clear - GA inhibits the process as a whole (14, 59, 65). The literature data show that high temperature, long day and the rich content of nitrates in nutrient medium promote the raise of gibberellins content in apical zone of stolons (42), which inhibits TB (65, 66). It was suggested (67) that the inhibitory effect of gibberellins on TB of potato depends on glucose content in the medium (the idea wasn’t proved by the authors).
Abscisic acid (ABA). The role of exogenous ABA in TB of potato is not completely clear. ABA inhibits stolon growth (27, 57), relieves apical dominance (28), regulates phytohormones balance and inhibits kinetin-induced TB (57). It is believed that ABA is not a trigger for TB (27, 28), but it only promotes the thickening of stolon apex (68). Studying the influence of GA / ABA ratio (against a background of 8% sucrose), it has been found that raising the concentrations of both phytohormones in a medium inhibits TB (68). ABA also inhibits rhizogenezis in stem explants (16), reduces TB efficiency (59), it contributes to the increase in size of tuber cells and transformation of sucrose into starch (69).
Ethylene. C2H4 is the important but hardly-controlled hormonal factor of TB in potato. In the first hours after cutting, stem explants intensively emit CO2 and C2H4, and absorb O2 (70). In the first 2 weeks of microtubers development, the intensity of ethylene synthesis remains still high, but later it falls (71). In tightly closed or parafilm-sealed vessels, stem explants formed stolons while TB was inhibited. The analysis of air composition in the culture vessels has revealed the accumulation of ethylene, whose adsorption initiated TB (59). This fact shows the importance of ventilation in culture vessels: the high ethylene content promotes stolons formation, and removal the gas induces TB (72). Exogenous introduction of ethylene into a culture vessel via a silicon filter caused stolon apex to thicken without a consequent TB (73). Ethylene can inhibit TB and root growth as well, while the introduction of kinetin and carbon dioxide (74) or potassium permanganate into the culture vessel relieved the inhibitory effect of ethylene (59). Thus, under in vitro conditions, ethylene slows down the formation of stolons (which makes possible the initiation of TB), and at the same time ethylene inhibits TB process itself.
Retardants. Retardants are also named the stabilizers of stem growth (47). These synthetic compounds inhibit gibberellins biosynthesis in plants and slow the rate of stem growth (75). For example, the retardant chlorcholinechloride (CCC) against a background of 6% sucrose increases TB efficiency (50). The inhibitory effect of high temperature (28 °C) was completely removed after adding of CCC to a nutrient medium (76). The increase in CCC content in medium from 250 to 500 mg/l accelerated TB and leveled the time required for tubers formation in varieties of different ripeness groups (77). The combined effect of CCC and 6-BAP against a background of 6% sucrose stimulated TB as well (59). The introduction into a nutrient medium of ABA, CCC and coumarin in concentrations of 3, 100 and 25 mg / l, respectively, increased TB efficiency (78). The optimal for TB concentration of coumarin - 25-50 mg/l (79), and its restriction leads the formation of small microtubers. The tubers, induced by coumarin, were larger than those in the medium with kinetin, and TB started on the 10-14-th days after bringing plants into the medium with coumarin, which is 2-3 days earlier than in the medium with kinetin (79).
2,3-dihlorizobutirat sodium (DHIB) at a concentration of 100 mg/l stimulated TB (80), while DHIB is 10-20 times less toxic than CCC (47). Paclobutrazol inhibited stem growth, reduced levels of endogenous gibberellic acid by 60% (81), increased TB efficiency and / or weight of microtubers (63, 65). The effect of triazoles (Triadimefon, Uniconazole) has been studied in TB-problematic potato varieties (60). Even the low concentrations (0.01 mg/l) of these preparations increased the number and size of microtubers, compared with those induced by 6-BAP (10 mg/l); the best results were obtained from Uniconazole at a concentration of 0,5 mg/l. The similar result was observed after applying Tetcyclacis (TB recorded on the 4th day (82). Screening of other retardants (Chlormequat, Daminozide, Ancymidol) also showed their stimulating effect on TB (83).
The high TB rates were observed at the combined action of kinetin and CCC, as well as the larger microtubers compared with control (without growth regulators) (84). According to other authors (85), on the contrary, the nutrient medium with 8% sucrose caused the greater weight of tubers than that in the medium containing kinetin and CCC in addition to sucrose. The combined application of kinetin and CCC against a background of increased level of sucrose resulted in appearance of TB early signs on the 2nd day after the induction, and the more expressed process - from 5-th to 15-th day (86). The introduction of growth regulators or their analogues - the retardants CCC and coumarin - into a nutrient medium keeping the ordinary (not high) content of sucrose, did not induce TB (85).
From the first reports on obtaining of potato tubers in vitro, this matter still remains of an interest. This fact is explained by the use of microtubers as the unique model for studying physiological and biochemical processes, and also the current need in healthy seed grown by industrial way in vitro. Upon the studied data, we conclude that none of the considered factors (high content of sucrose, growth regulators, photoperiod, etc.) separately induces TB in vitro, but the process is stimulated by a complex of agents, including both endogenous (the ratio of phytohormones at a current ontogeny stage) and exogenous factors (photoperiod, temperature, etc.).
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K.A. Timiryazev Institute of Plant Physiology,