ISSN 10623590, Biology Bulletin, 2014, Vol. 41, No. 6, pp. 512–521. © Pleiades Publishing, Inc., 2014. Original Russian Text © M.R. Khaliluev, L.R. Bogoutdinova, G.B. Baranova, E.N. Baranova, P.N. Kharchenko, S.V. Dolgov, 2014, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2014, No. 6, pp. 586–596.
PLANT PHYSIOLOGY
Influence of Genotype, Explant Type, and Component of Culture Medium on in vitro Callus Induction and Shoot Organogenesis of Tomato (Solanum lycopersicum L.) M. R. Khalilueva, b, L. R. Bogoutdinovaa, b, G. B. Baranovaa, E. N. Baranovaa, P. N. Kharchenkoa, and S. V. Dolgova, c a
AllRussia Research Institute of Agricultural Biotechnology, ul. Timiryazevskaya 42, Moscow, 127550 Russia b Russian Timiryazev State Agrarian University, ul. Timiryazevskaya 49, Moscow, 127550 Russia c Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry (Pushchino Branch), Russian Academy of Sciences, pr. Nauki 6, Pushchino, Moscow oblast, 142290 Russia email:
[email protected] Received November 5, 2013
Abstract—The influence of explant type as well as of the type of growth regulators and concentration on cal lus induction and somatic organogenesis of shoots was studied in vitro on four tomato genotypes of Russian breeding. Cytological study of callus tissue was conducted. It was established that tomato varieties have a sub stantially greater ability to indirect shoot organogenesis compared with the F1 hybrid. The highest frequency of somatic organogenesis of shoots, as well as their number per explant, was observed for most of the geno types studied during the cultivation of cotyledons on Murashige–Skoog culture medium containing 2 mg/L of zeatin in combination with 0.1 mg/L of 3indoleacetic acid. An effective protocol of indirect somatic orga nogenesis of shoots from different explants of tomato varieties with a frequency of more than 80% was devel oped. DOI: 10.1134/S1062359014060041
INTRODUCTION Tomatoes are an important crop, which occupies the first place in the world among products of vegeta bles growing on its importance and the volume of pro duction, as well as the second place after citrus crops on a vitamin value. The global gross yield of marketable prod ucts of tomatoes in 2012 amounted to 161.8 million tons, Russia accounts for about 2.5 million tons (~1.5%) on a cultivation area of 117700 hectares (FAO, 2014). Tomatoes are widely used as a model object in various fundamental and applied research, for example, in the development of haploid and transgenic plants, based on the cell and tissue culture procedure in vitro. One of the main conditions of applying the tech nique of in vitro cultivation of isolated organs, tissues, and cells of tomato is the availability of highly effective protocols of producing valid fertile regenerants. Regeneration of tomato shoots from somatic cells is most often achieved by organogenesis with the previ ous stage of callus tissue formation (Bhatia et al., 2004; Jabeen et al., 2005; Rasid and Bal, 2010). Other meth ods, such as direct organogenesis of shoots or somatic embryogenesis, are used much less frequently (New man et al., 1996; Chandel and Katiyar, 2000). Morphogenesis in tissue culture is a complex pro cess, and its regulation is carried out on the cellular,
tissue, and organism levels (Butenko, 1999). The implementation of the morphogenetic potential of somatic cells is affected significantly by the genotype, physiological age, and origin of the explant, the quan titative and qualitative composition of plant growth regulators, the conditions of cultivation, and many other factors (Gubis et al., 2003; Ashakiran et al., 2011; Zhang et al., 2012). It has been established that the ability of various somatic tissues to undergo in vitro morphogenesis is under genetic control. It has been shown previously for many plant species, including representatives of the family Solanaceae (Yezhova, 2003; TrujilloMoya et al., 2011). It is known that wild Solanum species have a higher regenerative capacity than a cultivated tomato (Lech et al., 1996). At the same time the fre quency of shoot organogenesis in varieties and lines of tomato used to produce F1 hybrids is usually signifi cantly lower than that in model genotypes that are not economically valuable and are not used in the breeding process (Lima et al., 2004). Protocols for shoot organ ogenesis were currently developed for a number of for eign varieties and tomato lines with different regener ation frequencies. Cotyledons (Bhatia and Ashwath, 2008; Zhang et al., 2012), segments of epicotyl (Gubis et al., 2003), hypocotyl (Shahriari et al., 2006; Rashid and Bal, 2010), stem (Harish et al., 2010), and pedun
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INFLUENCE OF GENOTYPE, EXPLANT TYPE, AND COMPONENT Table 1. Type and concentration of plant growth regulators included in the culture medium for the induction of tomato callus formation and shoot organogenesis Plant Concentration of plant growth regulators, mg/L growth MS3 MS4 MS5 MS6 MS7 regulator MS2 Zeatin 6BAP IAA
1
1
2
– 0.1
– 0.5
– 0.1
– 2 0.1
– 4 0.1
1 2 0.1
6BAP—6benzylaminopurine, IAA—3indoleacetic acid; “–”—no component, for Tables 1–4.
cles (Compton and Veilleux, 1991); fragments of leaves (Chandel and Katiyar, 2000; Afroz et al., 2009), meristem (Mirghis et al., 1995), protoplasts (Gleddie et al., 1989), and immature embryos were used as explants (Young et al., 1987). For the induction of cal lus formation and shoot regeneration, explants were cultured on nutrient media with a wide range of types and concentrations of cytokinins (6benzylaminopu rine (6BAP), zeatin, kinetin, 2isopenteniladenin, or thidiazuron) in combination with a low concentration of auxins (3indoleacetic (IAA), 1naphthylacetic, or 2,4dichlorophenoxyacetic acids) or without them (Bhatia et al., 2004; Khaliluev et al., 2010, Osman et al., 2010). It should be noted that the morphogenic potential of cultured tissues reduces substantially in the process of genetic transformation. The reasons for this are direct infection with the pathogen (for Agrobacterium mediated transformation) or mechanical damage to plant tissue (for ballistic transformation), the inhibi tory effect of a selective agent, prolonged culturing under in vitro conditions, and other stress factors. In this regard studies of the regenerative capacity of a par ticular genotype and type of explant are needed, as well as selection of the cultivation conditions provid ing the output of the greatest amount of regenerants, the main one among them being the composition of the nutrient medium. The aim of research is to study the influence of tomato genotype, type of explant, and concentration of growth regulators included in the nutrient medium on the processes of morphogenesis in vitro, as well as the development of effective protocol of tomato shoot organogenesis based on commercial genotypes of Russian breeding. MATERIALS AND METHODS The seeds of commercial tomato varieties Rekords men, Prazdnichnyi, and Trans novinka bred in the All Russia Research and Development Institute of Irriga tion Vegetable and Melons RAAS (Astrakhan region) and the F1 hybrid Chernysh, originated at Michurin BIOLOGY BULLETIN
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skii State Agrarian University (Tambov region), were used as the plant material for research. To obtain donor tomato plants, seeds were surface sterilized for 10 s in 96% ethanol, and then for 7–8 min in 20% aqueous solution of the commercial chlorine bleach ACE containing a few drops of Tween 20. After sterilization, seeds were washed 3–4 times with sterile distilled water and placed in culture vessels with the nutrient medium based on mineral compo nents with vitamins prepared by the Murashige– Skoog medium (MS) (Murashige and Skoog, 1962) with the addition of 3% sucrose and 0.7% agar. Cotyledons, including 2 to 3mmlong petioles, and hypocotyl segments 10–15 mm in size, obtained from the middle part of 10 to 12dayold aseptic seedlings, were used as explants. For the induction of morphogenesis responses, the explants were cultured on medium without plant growth regulators (MS1), and with the addition of different concentrations of zeatin (Sigma, United States) and 6BAP (Sigma) in combination with IAA (Sigma) (MS2–MS7) (Table 1). Each experimental variant consisted of four replicates, and each of them had ten explants. Before autoclaving, the pH of culture mediums was adjusted to 5.7–5.8 with 1 M KOH. Sterilization of the culture medium was carried out by autoclaving for 20 min at 121°C and a pressure of 1.1 atm. The growth regulators were sterilized by passage through nitrocel lulose filters with a pore diameter of 0.22 µm (Milli pore, United States) and added after autoclaving to the medium cooled to 45°С. Cotyledon and hypocotyl explants were placed on the surface of the medium, with the horizontal and abaxial sides respectively. Sub culturing on fresh medium was carried out every 15 days. Cultivation of donor plants and explants was performed in an illuminated room with the following conditions: temperature 23–25°С, 2.5–3.0 klux illu mination, photoperiod 16/8 h (day/night). The effectiveness of morphogenesis responses was evaluated after 45 days of cultivation based on the fol lowing parameters: frequency of callus formation, fre quency of shoot organogenesis, and average number of shoots per explant. The frequency of callus formation, expressed as a percentage, was defined as the ratio of the number of explants that formed callus tissue to their total number. The frequency of shoot organogen esis, expressed as a percentage, was defined as the ratio of the number of explants in which shoot organogene sis occurred to the total number of explants that formed a callus. The average number of shoots per explant was determined as the ratio of the total num ber of regenerants to the explants in which organogen esis of shoots was observed. To perform light microscopy, fragments of hypo cotyl and cotyledon explants and callus tissue were fixed in 2.5% solution of glutaraldehyde (Merck, Ger
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KHALILUEV et al.
many) at 0.1 M Sorensen’s phosphate buffer (pH 7.2) with the addition of 1.5% sucrose. After washing off the fixing mixture, plant material was postfixed with a 1% solution of osmium tetroxide (ОsО4) (Sigma), dehydrated in ethanol with an increasing concentra tion (30, 50, 70, 96, and 100%), then in propylene oxide (Fluka, Germany) and embedded in a mixture of epoxy resins Epon812 and Araldita (Merck) according to standard protocol (Weekly, 1973). Semifine, 1 to 2micronthick sections were pre pared using an ultramicrotome LKBV (LKB, Swe den), mounted on glass slides and stained with 0.1% aqueous solution of methylene blue (Merck). Stained preparations were embedded in a mixture of epoxy resins. Preparations were analyzed and photographed under an Olympus BX51 microscope (Olympus, Japan) equipped with a Color View II camera (Soft Imaging System, Germany). Statistical analysis of the experimental results was performed using Student’s, Fisher’s, and Duncan’s tests with the program AGROS (version 2.11). The values, expressed as a percentage, before the analysis of variance were transformed using the function angle–arcsine X , where Х is the frequency of callus formation and shoot organogenesis. The values of the average number of shoots per explant before the anal ysis of variance were transformed with the formula X + 1, where X is the average number of shoots. RESULTS AND DISCUSSION It is known that an important condition for the dif ferentiation of plant cells and the formation of callus tissue is the presence of two classes of plant growth regulators, auxins and cytokinins, in the medium (Butenko, 1999). Due to this, we used six different cul ture mediums composed based on MS medium and with the addition of various types and concentrations of plant growth regulators (see Table 1). Morphogenic callus formation depends also on the type of explant and on the stage of its development. At the same time, its origin, physiological age, and size are essential too. It is believed that young, poorly dif ferentiated tissues containing an increasing number of competent cells have a morphogenetic ability (Chan del and Katiyar, 2000; Bhatia et al., 2004). Further more, the capability of regenerated shoots for rooting decreases substantially with an increasing age of the primary explant. We used cotyledons and hypocotyls derived from 10 to 12dayold seedlings. An increase in cell size was observed during the cul tivation of both types of explants on all variants of cul ture mediums at the beginning of the first passage. Thus, on the 5th day of cultivation thickenings in the form of overgrown tissues formed on the explants on which callus tissue formed subsequently. It is known
that cells relatively small in size, with dense cytoplasm, small vacuoles, and the nucleus located in its central part have the greatest ability to move into the differen tiated state (Butenko, 1999; Kruglova et al., 2007). As a result of cytological study, it was found that, despite the use of cotyledons and hypocotyls derived from juvenile plants for the experiment, the majority of cells from the epidermal and mesophyll layers had a strong vacuolization, and the cytoplasm and cellular com partments were displaced towards the peripheral part and located predominantly along the cell wall. Based on this, proliferating cells of the vascular bundle are a source of occurrence of callus tissue in both types of tomato explants (Figs. 1a, 1b). The formation of callus tissue on cotyledon leaves occurred on the cut of the petiole as well as on the abaxial surface of the leaf blade, whereas on the hypocotyl segments, it occurred on the entire surface of the explant. Callus tissue was predominantly of light green or yellowgreen color and of dense grain structure (Figs. 1c, 1d). According to the results of a threefactor analysis of variance, significant differences were found at the 5% level both in the frequency of callus formation between different genotypes and types of explants, and between the studied variants of culture mediums. Furthermore, the differences were significant for the interaction of the factors variant of the culture medium × genotype, culture medium × explant, and the genotype × explant, and for all three factors (Table 2). It was shown that the addition of plant growth reg ulators to the culture mediums stimulated the induc tion of morphogenesis in both types of explants. Dur ing the cultivation of cotyledonous leaves of all three genotypes on a culture medium with the addition of various types and concentrations of cytokinins in combination with IAA, the frequency of callus forma tion was maximal (100%), whereas no formation of the callus occurred in a medium without plant growth regulators. Necrosis of tissues occurred upon their subsequent cultivation. The frequency of callus formation was 47.5 and 32.4%, respectively, during the cultivation of hypo cotyl segments of tomato varieties Rekordsmen and Trans novinka on the medium without plant growth regulators, unlike that of cotyledonous leaves (Fig. 2). This indicates a high content of endogenous phyto hormones in explant tissues. The frequency of callus formation exceeded 95% during the cultivation of hypocotyl segments of the studied tomato genotypes in most variants of culture mediums containing plant growth regulators. An exception was the MS2 variant of the culture medium, containing 1 mg/L of zeatin in combination with 0.1 mg/L of IAA during the cultivation of hypocotyls of the tomato variety Rekordsmen. Significant differ ences were marked also in the frequency of callus for BIOLOGY BULLETIN
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(а)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
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Fig. 1. Callus formation and somatic organogenesis of tomato shoots in vitro. (a, b) a cross section of the middle segment of the cotyledonous leaf and hypocotyl, respectively. Scale: 50 microns. (c, d) formation of callus tissue on the explants of cotyledons and hypocotyls, respectively. (e, f) formation of meristematic loci in callus tissue, obtained from the explants of cotyledons (e) and hypocotyls (f). Scale: 50 microns. (g, h) mass indirect shoot organogenesis of the tomato variety Rekordsmen during the cul tivation of explants of cotyledons (g) and hypocotyls (h) on MS culture medium with the addition of 1 mg/L of zeatin and 0.1 mg/L of 3indolylacetic acid. BIOLOGY BULLETIN
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Table 2. The effect of tomato genotype, type of explant, and culture medium composition on the frequency of callus for mation in vitro Variance
ss
General Of variants Of factors A (medium composition) B (explant) C (genotype) Interactions AB AC BC ABC Errors
df
ms
F05
F
188978.02 183895.73
223 55
– 3343.56
– 1.18
– 110.53
159431.33 1110.55 1064.18
6 1 3
26571.89 1110.55 354.73
2.16 3.9 2.66
878.36 36.71 11.73
6419.37 7402.98 1064.18 7403.15 5082.28
6 18 3 18 168
1069.9 411.28 354.73 411.29 30.25
2.16 1.71 2.66 1.71 –
35.37 13.6 11.73 13.6 –
ss—sum of squares, df—degree of freedom, ms—mean square, F05—Fvalue at significance level of 0.05, F—actual value of Fisher’s test.
Frequency of callus formation, %
mation from hypocotyl explants of the varieties Prazd nichnyi and Trans novinka during cultivation on the MS4 and MS5 mediums, respectively. Furthermore, the simultaneous presence of two types of cytokinins in the culture medium had a negative effect on callus formation from hypocotyl explants of these genotypes. The average frequency of callus formation from hypo cotyls explants of the varieties Trans novinka (78.9%) and Prazdnichnyi (81.2%) was significantly lower than that of the variety Rekordsmen (89.4%) for all variants of culture mediums. The callogenesis ability of cotyle don explants was significantly higher than the similar ability of hypocotyls segments in all studied genotypes. The results of cytological analysis indicate that cal lus tissue formed from explants of cotyledons and
hypocotyls was characterized by a significant variabil ity of cells in size and shape (Figs. 1e, 1f). In general, they can be classified into two morphological types— meristematic and parenchymal. The cells of the mer istematic type were located in the callus tissue as large local accumulations, forming meristematic loci. According to the literature, meristematic cells are actively proliferating (Kruglova et al., 2007). They are characterized by a relatively small size and contain dense cytoplasm, small vacuoles, and large nuclei located mainly in the central part of the cell. Callus cells of the parenchymal type are large in size and have a small amount of cytoplasm and a welldeveloped system of vacuoles in which, as we know, an accumu lation of secondary metabolites occurs (Butenko,
100 80 60 40 20 0 MS1
MS2
MS3
MS4 1
MS5 2
3
MS6
MS7
Mean
4
Fig. 2. Effect of exogenous plant growth regulators on the frequency of callus formation in hypocotyl segments of tomato geno types. (1–4) genotypes of tomato varieties Rekordsmen, Trans novinka, Prazdnichnyi, and F1 hybrid Chernysh, respectively. BIOLOGY BULLETIN
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1999; Seldimirova and Kruglova 2013). A high degree of cell vacuolization is an indicator of retardation of their growth and aging. Parenchymal cells are round, oval, or elongated, settling mainly around the mer istematic locus. The meristematic loci of callus tissue formed from cotyledonous leaves had a dense uniform structure with a distinct abaxial and oblique–abaxial orienta tion of cell divisions. Unlike meristematic cells of cal lus tissue obtained from cotyledons, cells of the mer istematic locus of callus tissue formed from hypocotyl explants were much smaller in size and were most likely formed by unequal periclinal and anticlinal divi sions. An increasing number of prismatic cells in the peripheral part of the morphogenetic locus may be noted, as well as isodiametric cells with dense cyto plasm in the inner area. It was possible to observe the arrangement of cells in rows or lacunas and to assume the presence of elements of the conduction system. Later, organogenesis of tomato shoots from morpho genetic loci of callus tissue occurred. In the experiment, callus formation and shoot organogenesis occurred on a culture medium of the same composition, which was also previously men tioned (Bhatia et al., 2004; Zhang et al., 2012). Depending on the type of cultivated tissue and the composition of the culture medium, the frequency of somatic organogenesis of tomato shoots varied from 21.8 to 100% (Table 3), and their average number per explant varied from 1.99 to 5.06 (Table 4). The highest frequency of shoot organogenesis was observed during the cultivation of cotyledonous leaves and hypocotyl fragments on culture mediums with the addition of 1 or 2 mg/L of zeatin in combination with 0.1 mg/L of IAA (Figs. 1g, 1h). It was found that the frequency of organogenesis and the average number of shoots per explant were significantly higher during the cultivation of both types of explants on culture mediums with zeatin compared to 6benzyladenine. Furthermore, shoot organogenesis began, on average, 3–5 days ear lier during the cultivation of explants on culture medi ums containing zeatin. Several authors noted a positive effect of two type of cytokinins on cell differentiation of tomato explants in vitro (Krasnyanski et al., 2001; Rashid and Bal, 2010). In our study, the simultaneous presence of two cytokinins in the culture medium did not stimulate the organogenesis, but, on the contrary, reduced signifi cantly the frequency of shoot organogenesis from cot yledon and hypocotyl explants as well as decreased their number compared to variants containing only zeatin in optimal concentrations. It was found that the frequency of shoot organo genesis of cotyledon explants was significantly higher than that of hypocotyl segments for all genotypes except the F1 hybrid Chernysh. By the frequency of BIOLOGY BULLETIN
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shoot organogenesis from cotyledon explants, tomato genotypes were ranged in the following order: Prazd nichnyi (70.2%) > Rekordsmen (57.5%) > Trans novinka (48%) > Chernysh (12.5%). Just as in the case of cotyledon explants, the F1 hybrid Chernysh was characterized by the lowest fre quency of shoot organogenesis from hypocotyl seg ments. At the same time, no significant differences were revealed on this value between the varieties Reko rdsmen, Trans novinka, and Prazdnichnyi. In general, the tomato variety Prazdnichnyi had the greatest mor phogenetic in vitro capacity according to the fre quency of shoot organogenesis (54.7%). No significant differences in the number of regen erating shoots were observed between the explants for most of the studied genotypes (Rekordsmen, Trans novinka, and Chernysh). A significantly greater num ber of shoots regenerated from callus tissue formed on cotyledon explants (3.37) was identified only for the variety Prazdnichnyi, compared to callus obtained from hypocotyl segments (2.09). At the same time, the average of shoots number formed on one cotyledonous leaf of the tomato variety Prazdnichnyi was the highest and exceeded significantly the corresponding value for cotyledon and hypocotyl explants of other genotypes. In general, by the number of regenerating shoots per explant, the studied genotypes were ranged in this order: Prazdnichnyi (2.73), Trans novinka (2.71) > Rekordsmen (2.32) > Chernysh (1.82). It was found that MS4, containing zeatin and IAA at concentrations of 2 and 0.1 mg/L, respectively, is the culture medium that ensures, on average, the greatest frequency of shoot organogenesis (68.8%) and their number per explant (3.19) for all studied geno types and explant types. Important indicators for regenerating shoots include not only their quantitative but also their qual itative characteristics. It is known that shoots regener ating from callus tissue may show signs of vitrification. Vitrification mechanisms are not entirely clear so far. Vitrification of shoots can be caused by exogenous growth regulators (auxins, cytokinins) and their imbalanced ratio, as well as by the high water potential of the culture medium (Ziv, 1991). Vitrification of shoots was noted during the cultiva tion of hypocotyl segments of the variety Rekordsmen on the MS7 culture medium, containing two type of cytokines, and of cotyledon and hypocotyl explants of all genotypes on the MS3 medium, characterized by a high content of auxins compared to other variants. Shoots regenerating from different explants were characterized by length (Fig. 3). In general, the length of shoots obtained from cotyledon explants cultivated in mediums with the addition of plant growth regula tors was higher than that from hypocotyl segments. Furthermore, regenerants formed from callus tissue
47.4 defg 26.4 cd
MS6
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48.6 b
39.6 c
28.1 cd
10 bc
88.3 ij
63 fg
59 efg
19.5 cd
13 bcd
87.7 j
–
48 d
45.5 klm 54.9 fgh
36.9 ijkl
27.5 ghi
79.3 tu
73.7 pqrst 80.5 hij
80.5 hij
0a
43b
38 c
46.9 efg
2.7 ab
24.2 de
90 j
71.4 ghij
29.7 def
1.3 ab
cotyle hypocotyls dons 0.7 ab
mean
0a
cotyle dons
–
50.9 lm
11.1 cdef
18.6 fghi
88.9 v
76 rst
70.2 f
83.2 gh
58 ef
54.9 ef
99.3 ij
96.2 hij
55.1 mn 100 j
Trans novinka
54.7 c
39.1 c
55.5 ef
28.7 cd
11.2 bc
56.5 ef
47.4 de
74.1 fg
0a
hypocotyls
mean
0a
hypocotyls
0.6 a
16.7 def
21.8 efg
16.7 def
7.4 bcd
22.3 efg
36 g
16.7 def
12.2 cde 32.1 fg
0a
cotyle dons
Chernysh
–
12.5 a
15.8 a
19.1 b
69.4 nopqrst 19.5 defg 19.5 defg
43.4 jklm
33.1 hijk
77.9 stu
71.8 opqrst
87.1 uv
0a
Prazdnichnyi
Variants labeled with same letters do not differ significantly by the Duncan’s test (α = 0.05) (for Tables 3 and 4). 1 The effect of genotype and culture medium composition on the frequency of tomato shoot organogenesis. 2 The effect of nutrient medium composition on the frequency of tomato shoot organogenesis. 3 The effect of explant type and culture medium composition on the frequency of tomato shoot organogenesis. 4 The effect of genotype and explant type on the frequency of tomato shoot organogenesis. 5 The effect of genotype on the frequency of tomato shoot organogenesis.
Mean (genotype)5
57.5 e
44.9 def
MS5
Mean (explant)4
70.2 ghi
MS4
62.9 fg
84.4 hij
MS3
MS7
92.5 j
MS2
75.8 qrst
1.2 ab
03 a
MS1
2.4 ab
mean1
Rekordsmen
cotyle hypocotyls dons
Culture medium
Frequency of shoot organogenesis, %
Table 3. Effect of genotype, explant type, and culture medium composition on the frequency of tomato shoot organogenesis in vitro
60.1 e
0.5 a
–
19.5 efg
4 bc
19.5 fg
28.9 ghi
–
–
46.3 c
23.9 b
24.7 b
68.8 f
16.7 defg 59.6 de
22.2 fgh
0a
mean
Mean (medium composi tion)2
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2.43 cde 2.19 c
MS7
2.32 b
2.32 bcd
2.19 c
3.42 fg
3.8 gh
1a
–
2.31 fgh
2.24 fgh
2.05 fg
2.85 d
2.71 c
2.57 cd
2.66 def 2.47 def
2.34 cde 1.44 ab
1.88 bcd 1.88 bcd
5.06 i
3.28 efg
2.62 def
1.21 ab
1a
2.47 de
3.88 gh
4.49 hi
–
2.57 hijk
3.37 e
3.65 fgh
1.89 cdef 2.66 de
1.88 def
4.77 q
3.35 no
3.21 mno 5.43 i
1.11 a
2.73 c
2.09 abc
2.5 de
1.99 cd
1.56 bc
2.13 cde
2.5 de
2.92 ef
1a
2
mean
2.07 d
1.99 d
1a
1.39 bc
2.07 d
–
1.8 a
3.08 klmno 2.1 d
2.33 fgh
2.02 ef
Chernysh
1.82 a
1.84 a
1.99 d
1.72 cd
1.99 d
2.13 d
2.07 d
1.99 d
1a
cotyledons hypocotyls
3.01 jklmno 1.99 d
3.5 o
4.18 p
1a
Prazdnichnyi
cotyle hypocotyls average cotyledons hypocotyls dons
2.91 ijklmno 4.88 hi
2.47 ghij
3.19 lmno
1.11 a
mean1
Trans novinka
The effect of genotype and culture medium composition on the average number of tomato regenerants. The effect of culture medium composition on the average number of tomato regenerants. 3 The effect of explant type and culture medium composition on the average number of tomato regenerants. 4 The effect of genotype and explant type on the average number of tomato regenerants. 5 The effect of genotype on the average number of tomato regenerants.
1
Mean (genotype)5
2.32 cd
2.28 cd
MS6
Mean (explant)4
2.1 c
MS5
1.99 bc
2.79 defg 3.03 efg
MS4
3.13 fg
1.21 a
2.43 cde 2.5 cde
3.24 g
13 a
cotyle hypocot dons yls
Rekordsmen
MS3
MS2
MS1
Culture medium
The number of shoots per explant
Table 4. Effect of genotype, explant type, and culture medium composition on the average number of shoots per explant of tomato in vitro
2b
3.19 f
2.85 d
3.14 ef
1.06 a
–
2.05 fgh
–
–
2.5 c
1.41 bcde 1.97 b
2.03 fg
2.06 fgh
2.07 fgh
1.99 fg
1a
mean
Mean (medium composi tion)2
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Regenerant length, cm
(а)
(b)
1 2
1
1
0
0 (d)
(c)
1
1
0
0 MS2 MS3 MS4 MS5 MS6 MS7
MS2 MS3 MS4 MS5 MS6 MS7
Culture medium Fig. 3. Morphometric characteristics of tomato shoots regenerated from callus tissue of cotyledon (1) and hypocotyl (2) explants of varieties Rekordsmen (a), Trans novinka (b), Prazdnichnyi (c), and F1 hybrid Chernysh (d).
during cultivation in mediums containing 6BAP had were less in length. CONCLUSIONS The experimental data show a significant depen dence of the efficiency of in vitro tomato shoot organ ogenesis on the genotype, explant source, and type and concentration of plant growth regulators included in the culture medium. It was found that the variety Prazdnichnyi had the greatest ability for indirect shoot organogenesis among the four tomato genotypes ana lyzed. The F1 hybrid Chernysh had a low regenerative capacity compared to known varieties of tomato (the frequency of shoot organogenesis and their number per explant did not exceed 29% and 2.1, respectively). It was shown that for the majority of studied genotypes of tomato, cotyledon explants were characterized by a greater ability of callus formation and of shoot somatic organogenesis compared to hypocotyl segments. The frequency of tomato shoot organogenesis in vitro, as well as their qualitative and quantitative characteristics, depends on the type and concentra tion of the growth regulators applied. The preferred use of zeatin compared to 6BAP was established. In general, for all examined tomato genotypes and type of cultivated tissues, the best culture medium providing maximum frequency of somatic shoot organogenesis
(68.8%) and their number (3.19) is the MS medium with the addition of 2 mg/L of zeatin in combination with 0.1 mg/L of IAA. Thus, an effective protocol of indirect shoot organ ogenesis from cotyledon and hypocotyl explants was developed on the basis of the Russian tomato varieties Rekordsmen, Prazdnichnyi, and Trans novinka, which can be used in genetic transformation experi ments. REFERENCES Afroz, A., Chaudhry, Z., Khan, R., et al., Effect of a GA3 on regeneration response of three tomato cultivars (Lyco persicon esculentum), Pak. J. Bot., 2009, vol. 41, pp. 143– 151. Ashakiran, K., Sivankalyani, V., Jayanthi, M., et al., Geno type specific shoots regeneration from different explants of tomato (Solanum lycopersicum L.) using TDZ, Asian J. Plant Sci. Res., 2011, vol. 1, pp. 107–113. Bhatia, P., Ashwath, N., Senaranta, T., and Midmore, D.J., Tissue culture studies of tomato (Lycopersicon esculentum), Plant Cell, Tiss. Org. Cult., 2004, vol. 78, pp. 1–21. Bhatia, P. and Ashwath, N., Improving the quality of in vitro cultured shoots of tomato (Lycopersicon esculentum Mill cv. Red Coat), Biotech., 2008, vol. 7, pp. 188–193. Butenko, R.G., Biologiya kletok vysshikh rastenii in vitro i biotekhnologii na ikh osnove (Biology of Higher Plant Cells in vitro and Biotechnology Based on Them), Moscow: FBKPress, 1999. BIOLOGY BULLETIN
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Translated by M. Shulskaya
