Sections of different explants (cotyledon, hypocotyl or meristem-type), 2,4-D (0.3 and 1.0 mgl-1 and kinetin (0 and 1.0 mgl-1) were examined in MS medium which was solidified with 0.8% Bacto-agar for callus induction of soybean.
2,4-D at a concentration of 0.3 mgl-1 with 1.0 mgl-1 kinetin was considerably better in all the explants for optimal callus growth than other hormonal concentrations.Postavljen je trofaktorijelan pokus (2x2x3) u 6 ponavljanja u svrhu ispitivanja utjecaja 2,4-D (0,1 i 0,3 mgl-1) i kinetina (0,0 i 1,0 mgl-1) te eksplantatskih izvora (kotiledone, hipokotil ili vegetacijski vršci) na kvalitetu i težinski prirast kalusa soje (Glycine max /L./ Merr, linije 146/82-5). Bodovne procjene kvalitete kalusa testirane su HI2-testom (Tab. 1). Ustanovljena je različitost bodovnih procjena po harmonskim kombinacijama u odnosu na pretpostavku da razlika u kvaliteti nema. Parcijalnim testiranjem parova (Tab. 2) izdvojen je kao kvalitetniji kalus na kombinacijama auksina s kinetinom nego bez kinetina. Istosmjerna ocjena je moguća analizom vrijednosti težinskih izmjera (Tab. 3) provođenjem F-testa i t-testa (Tab. 4). Optimalna kombinacija za težinski prirast kalusa je 0,3 mgl-1 2,4-D i 1,0 mgl-1 kinetina. Nije uočena statistički značajna razlika između kultiviranih kotiledonskih ili hipokotilskih odsje- čaka ili pak vegetacijskih vršaka

Renata Pavlina

English

Postavljen je trofaktorijelan pokus (2x2x3) u 6 ponavljanja u svrhu ispitivanja utjecaja 2,4-D (0,1 i 0,3 mgl-1) i kinetina (0,0 i 1,0 mgl-1) te eksplantatskih izvora (kotiledone, hipokotil ili vegetacijski vršci) na kvalitetu i težinski prirast kalusa soje (Glycine max /L./ Merr, linije 146/82-5). Bodovne procjene kvalitete kalusa testirane su HI2-testom (Tab. 1). Ustanovljena je različitost bodovnih procjena po harmonskim kombinacijama u odnosu na pretpostavku da razlika u kvaliteti nema. Parcijalnim testiranjem parova (Tab. 2) izdvojen je kao kvalitetniji kalus na kombinacijama auksina s kinetinom nego bez kinetina. Istosmjerna ocjena je moguća analizom vrijednosti težinskih izmjera (Tab. 3) provođenjem F-testa i t-testa (Tab. 4). Optimalna kombinacija za težinski prirast kalusa je 0,3 mgl-1 2,4-D i 1,0 mgl-1 kinetina. Nije uočena statistički značajna razlika između kultiviranih kotiledonskih ili hipokotilskih odsje- čaka ili pak vegetacijskih vršaka.Sections of different explants (cotyledon, hypocotyl or meristem-type), 2,4-D (0.3 and 1.0 mgl-1 and kinetin (0 and 1.0 mgl-1) were examined in MS medium which was solidified with 0.8% Bacto-agar for callus induction of soybean.

2,4-D at a concentration of 0.3 mgl-1 with 1.0 mgl-1 kinetin was considerably better in all the explants for optimal callus growth than other hormonal concentrations

Pavlina, Renata

Hrčak - Portal of scientific journals of Croatia

Acta Bot. Croat. 45, 63— 70, 1986. CODEN: ABCRA2 YU  ISSN 0365— 0588UDC 582.738:632.954 =  20T H E  E F F E C T  O F  2 , 4 - D  A N D  K I N E T I N  O N  S O Y B E A N  C A L L U S  C U L T U R E *RENATA PAVLINA  (Faculty of Agriculture, University of Zagreb)Received January 17, 1986Sections of different explants (cotyledon, hypocotyl or meristem-type), 2,4-D (0.3 and 1.0 mgl-1 and kinetin (0 and 1.0 mgl-1) were examined in MS medium which was solidi­fied with 0.8% Bacto-agar for callus induction of soybean.2,4-D at a concentration of 0.3 mgl-1 with 1.0 mgl-1 kinetin was considerably better in all the explants for op­timal callus growth than other hormonal concentrations.I n t r o d u c t i o nOne of the main problems of in vitro plant induction has been the finding of medium ( B o u l t e r  and C r o c o m o  1980) for each geno­type of leguminous members separately ( G r e s s h o p p  and M o h a -  p o r t a  1981). According to Reinert’s theory of organogenesis ( B o u l t e r  and C r o c o m o  1980), minimal quantitative changes in relations to de­termined components in media are a determining factor in stimulating the development and differentiation of some organs.Depending on the genotype examined scientists do not agree on the medium compositions used, the sources and quantités of carbon, the source of nitrogen (of organic and inorganic origin), as these differences are expressed even in organic and inorganic additions to media. There are also numberless differences in the use of expiants.Growth of soybean callus tissue has been tried on media of different micro- and macro-elements composition. P o 1 a c c o (1976) reports a suc­cessful cultivation of soya on MS basic media. Nutritive B5 medium has stimulated soya cells proliferation in experiments of many authors ( G a m b o r g  a n d S h y l u k  1970, B a y l e y  et  al. 1972, T r e s s h of f and M o h a p o r t a  1981, etc.). On modified Miller’s medium F o s k e t  and T o r r e y  (1969) achieved callus induction, and O s w a l d  e t a l .  (1977) point out Phillip’s medium as very favourable for callus growth.The success and favourability of soya culture growth depended upon a just selection of organic additives, hormones particularly. Key hor­* This paper is dedicated to prof. Z. Dévidé on the occasion of his 65th birthday.A C T A  B O T . C R O A T . V O L . 45, 1986. 63R E N A T A  P A V L IN Amones were primarily of auxine and cytokinine. 2,4-D solution concen­tration ratio varied from 1 to 22 p.Ml-1 ( F o s k e t  and T o r r e y  1969, G a m b o r g  and S h y l u k  1970, B a y l e y  et al. 1972, C hu  and L a r k  1976, C o l l i n s  e t a l .  1978, G o s a l  and B a j a j  1979, etc.). The variation grade of kinetin concentrations used ranged from 0.0 to 2.5 [iMl-1 ( B l a y d e s  1966, W it  h am  1968).Quantity differences were evident in the source of carbon used (saccharose) in the breeding of legumes in vitro of 20 gl-1 ( B a y l e y  et al. 1972) to 60gl-1 ( P e v a l e k  1979).Photoperiod lasted from 12 ( Ev a n s  et al. 1979) to 24 hours ( Wi t h am 1968).Since chemical and physical conditions differ in cultivation de­pending on the genotype used, the need for testing optimum culturing conditions for each genotype individually is becoming increasingly im­portant.The aim of the experiment was to select positive factors for callus in vitro of high protein soya genotype 146/82-5, by varying the breeding conditions. I have studied separately callus induction in relation to growth hormones (kind, concentration and combination) as well as ex­plant sources.Stimulation of soybean callus in vitro was the first step in creating conditions for determining urea metabolism in vitro in cell suspension culture. It was taken as the base for selection programme of urease- -overproducing mutants separation. Since urease has higher methionine levels, concentration increase improves seed protein quality by plant cell culture technique.M a t e r i a l s  a n d  M e t h o d sSoybean (Glycine max. /L./ Merr., genotype 146/82-5) was left swel­led in a knitted sack for 24 hours in running water. Subsequently, it was sterilized in 96% alcohol by a single washing. A 15-minute sterilization in blending machine followed (3% solution of IZOSAN — Pliva Zagreb). The treatment was finished by 4 successive 10-minute washings with sterilized destilled water. • ■The seeds were left germinating in Petri dishes on sterile moist cot­ton with 12-hour photoperiod, 1600 lx  light intensity, at a temperature of 20 "C.Aseptic technique was applied to explant in vitro culturing. Thirteen- -day old seedlings were taken for the experiment (3x2x2); they arranged by block-method, 6 repetitions at 24 °C, 1500 lx  and 16-hour photoperiod.A combination of micro- and macro-elements ( M u r a s h i g e  and S k o o g  1962) was utilized, varying the following 3 factors:1. Source of explant with 3 factor levels: ext — shoot tips (4.0 ±  1.2 mg)ex2 — hypocotyl sections (23.0 ±  5.9 mg) ex„ — cotyledon sections (58.0 ±  12.4 mg)2. Auxin (2,4-D) in concentrations 0.1 mgl-1 and 0.3 mgl"13. Cytokinin (kinetin) in concentrations 0.0 mgl-1 and 1.0 m gr1.A two-month growth of fresh matter (in grams), visual, descriptive and score estimation of callus by Stereo-microscope inspection, were indicators of a determined and examined combination effectiveness.64 A C T A  B O T . C R O A T . V O L . 45, 1986.TH E  E F F E C T  O F K IN E T IN  ON  S O Y B E A N  C A L L U S  C U L T U R EThe results of weight measurements were F and t-tested in relation to the analysis of variance of trifactorial experiment 3x2x2, and the block-method ( S n e d o c o r  and C o c h r a n  1967), though visual eva­luations were given in scores scale of 1—4 (Table 1), was tested with non-parametral HP-test considering Yates’ correction of measuring re­sults ( Pe t z  1981).R e s u l t s  a n d  D i s c u s s i o nHormonal combinations 2,4-D without kinetin especially in shoot- -apical meristems, resulted in watery, granular, transparent and spora­dic necrotic callus.Well-grown, watery, granular, and greenish callus without necrosis developed in both auxin concentrations, in the presence of kinetin (1 mgl-1), particularly in hypocotyl and cotyledon explants. This descrip­tive evaluation conforms with the estimation score, shown in Table 1.The results shown in Table 1 were HP tested with Yates’ result correction ( Pet z  1981). In hormonal concentration combinations (HP =  =  33.158**) differences in score estimations of callus growth were signi­ficant. Partial HP-tests for each pair of hormonal combinations were separately calculated (for a total of 6 pairs — Table 2) for the determi­nation of combinations showing significant difference.Results of the experiment allow a conclusion of kinetin utilization feasibility. All combinations without kinetin were poor and insufficient in respect of the score estimation and descriptive evaluation.Differences in estimations of callus growth between auxine concen­trations (0.1 or 0.3 mgl-1 2,4-D) are not statistically significant.Analysis of callus weight growth measurements by the method of preparatory variance analysis, proved that the greatest part of dispersive processes were caused by the differences in combinations (Fexp =  27.59**).Testing (F-test) of some component combinations (factors and in­teractions) showed that the fresh weight increase was independent of explant source, however, greatly dependent upon the presence of 2,4-D (factor B) kinetin (factor C). and their mutual presence in medium (em­phasized by the justified interaction BxC — Table 3).In order to determine differences in medium weight growth in view the combinations, t-test was carried (Table 4).The medium composition of 0.3 mgl-1 2,4-D and 1 mgl-1 kinetin showed evident advantages in comparison to other hormonal combina­tions.Equal growing possibilities were provided for all explant sources, and the experiment response was callus growth equally sufficient in all explant sections regardless of the origin.Therefore the utilization of any of the above mentioned parts of the plant is made possible with high prospects for positive (and equal) reactions in vitro. This is worth mentioning as it offers greater possibi­lity of employment of a plant as the explant source, because it reduces the need for breeding area.The results of my experiment are in conformity with W i t h a m ’ s results (1968), who succeeded, by cultivating cotyledon segments (cv. Acme), in inducing callus growth on Miller’s medium. He found the combined effect of 2,4-D and kinetin as more effective than 2,4-D alone.A C T A  E O T . C R O A T . V O L . 45, 1985. 65R E N A T A  P A V L IN ATable 1. Estimations of callus growth on MS-mediumExplant2.4 — D +  kin Estimation Combi­nationsm gl"1 0 + 1 + 2+ 3 +0.3 +  1 0 0 1 5 6K 0.3 + 0 0 6 0 0 6cotyledon 0.1 +  1 0 0 5 1 60.1 +  0 3 3 0 0 6cotyledon callus: 3 9 6 6 240.3 +  1 3 0 2 1 6H 0.3 + 0 3 3 0 0 6hypocotyl 0.1 +  1 1 1 0 4 60.1 + 0 3 0 0 3 6hypocotyl callus: 10 4 2 8 240.3 +  1 0 0 0 4 4VV 0.3 +  0 0 4 0 0 4meristem tip 0.1 +  1 2 2 0 0 40 . 1 + 0 1 2 0 1 4meristem-tip callus: 3 S 0 5 16ESTIMATIONS: 16 21 8 19 64H I  (horm on com binations) “  33.158 n—1 =  (4 -1 ) ( 4 - D  = 9H I 2 (explants) =  8 .9 4 8 n—1 =  (3 -1 ) ( 4 - D  = 6+  =  no growth1 =  callus dark brown, only very slight growth2 =  callus watery, glassy, greenish, good growth3 =  callus watery, glassy, greenish, very good growthTable 2. Summary of all testing results on MS-medium (HI2 for all pairs of hormonal combinations were tested on MS-medium)(2 .4—D  + k in /m g l- 1 ): (2 .4—D  +  kin /m gl-  2) n -  1 H I 2 (experi mcntal)(0.3 +  1) : (0.3 +  0) (2 — 1) (4 — 1) =  3 21 .47++(0.3 +  1) : (0.1 +  1) (2 — 1) (4 — 1) =  3 2.02(0.3 +  1) : (0.1 +  0) (2 — 1) (4 — 1) =  3 9.83 +(0.3 +  0) : (0.1 +  1) (2 — 1) (4 — 1) =  3 1242++(0.3 +  0) : (0.1 +  0) (2 — 1) (4 — 1) =  3 5.51(0.1 +  1) : (0.1 +  0) (2 -  1) (4 -  1) -  3 8.26+H I  (th eoretica l) ' 11*341 H I 2 (th eoretica l) =  7.815+  +  (p 0,01) +  (p 0,05)66 A C T A  B O T . C R O A T . V O L . 45, 1986.TH E  E F F E C T  O F K IN E T IN  ON  S O Y B E A N  C A L L U S  C U L T U R ETable 3. Table of variance analysis of experimentSource of variability SQ n—1 F „pTotal 50.74 47Block 0.61 3Comb 45.22 11 4.111 27.59+ +Mistake 4.91 33 0.149Factor A 0.79 2 0.40 2.68Factor B 3.89 1 3.89 26.10+ +Factor C 33.11 1 33.11 222.21+ +A x B 0.96 2 0.48 3.22A x C 0.03 2 0.02 0.13B x C 5.54 1 5.54 37.18 + +A x B X C 0.91 2 0.46 3.09Table 4. Average growth of fresh weight for soybean calus, on MS-medium with three explants stock and four hormonal combinations 2,4—D and kinetinExplants 2 ,4 -D  +  kin (mgl-1 )X(g)X(g)0.3 + 1 13.20 2.20K 0.3 + 0 4.23 0.71Cotyledon 0.1 + 1 7.01 1.170.1 + 0 2.96 0.490.3 + 1 12.18 2-03H 0.3 + 0 1.45 0.24Hypocotyl 0.1 + 1 5.88 0.980.1 + 0 3.51 0.590.3 + 1 10.45 2.61VV 0.3 + 0 2.06 0.52meristem 0.1 + 1 7.95 1.99shoot-tip 0.1 + 0 2.59 0.65GD, 0,01 =  0.44 GDp 0,05 =  0.32A C T A  B O T . C R O A T . V O L . 45. 1986. 67R E N A T A  P A V L IN AThe same effect could be achieved with a lower concentration of auxin in the presence of kinetin. However, high concentrations of 2,4-D could provoke kinetin to react as inhibitor. Optimal concentration ratio should be established henceforth, for callus induction in the presence of both hormones.Since cytokinin with slight effect, as adenine, methyladenine or ethyladenine, requires the presence of auxin ( M i l l e r  1967, according to W i t h a m 1968) in higher concentrations than in strong cytokinin, a synergistic effect of two hormonal groups could be expected.In my experiment the interaction of auxin and cytokinin was statis­tically justified; it is one proof more of the exactness of Miller’s obser­vations.The testing of differences in score estimations between pairs of combinations, proved lower concentrations of 2,4-D with kinetin, to be higher graded than the higher auxin concentrations without kinetin; however, there was no difference in the score estimation between two auxin concentrations without kinetin in the medium. This observation is in conformity with Miller’s hypotesis of synergism between these two groups of hormones.It seems that auxin reactions appear critical for the efficiency of cell division; therefore, to learn more about the possibilities of influencing the process has become very important.The efficiency of cell division could be increased by metabolic-mo­difications caused by cytokinins or by employment of more effective auxin (W i t h a m 1968), but also by utilization of certain combinations of inorganic salts, which could induce a physiological process of auxin synthesis unknown to us, excluding the need for auxin addition to the medium ( B r a u n  and W o o d  1962).Scientific interpretations can be reduced to the sole conclusion, namely that a cell without auxin cannot have optimum mitotic activity, and that the efforts to find favourable auxin concentrations or syner­gists in this activity or activators in vitro auxin syntheses, lead to a common goal: provoking the cells to intensified multiplication.These results may cotribute to a better understanding synergistic auxin and cytokinin relations.C o n c l u s i o nThe scope of the experiment was to find favourable auxin and cyto­kinin combinations, and differentiation of the value of explant sources for soybean callus induction.The type of explant (cotyledon, hypocotyl or shoot-tip) has not revealed statistically significant difference in intensity and quality of callus induction. According to score estimations of the quality and fresh weight measurements, the optimum hormonal combination was the con­centration of 0.3 mgl-1 2,4-D and 1.0 mgl-1 kinetin. Presumably, a range- -correlation between score estimations and callus measurements would be worth establishing, which would emphasize the prognostic value of visual evaluation, and since it is technically faster to perform, in case of good prognostic value, it could be readilly acceptable.•68 A C T A  B O T . C R O A T . V O L . 45, 1986.TH E E F F E C T  O F K IN E T IN  ON  S O Y B E A N  C A L L U S  C U L T U R EThe experimental part of this investigation was made in the Laboratory for Plant Cell Culture in the Department of Botany of the Faculty of Science, University of Zagreb.I thank Dr. Sibila J e 1 a s k a for her help and sugestions.R e f e r e n c e sBayley, J. M., J. King, O. L. Gamborg, 1972: The ability of amino compounds and conditioned medium to alleviate the reduced nitrogen requirement of soybean cells grown in suspension cultures. Planta (Berl.) 105, 25— 32.Blaydes, F. D., 1966: Interaction of kinetin and various inhibitors in the growth of soybean tissue. Physiol. Plant. 19, 748— 753.Boulter, D„ O. J. Crocomo, 1980: Plant cell culture implications: Legumes. In: Plant Cell and Tissue Culture: Principles and Applications. (Ed.: W. R. Sharp, P. O. Larsen. E. F. Paddock, V. Raghavan), 615— 631, Ohio State Univ. Press, Columbus.Braun, A. C., H. N. Wood, 1962: On the activation of certain essential bio­synthetic svstems in cells of Vinca rosea L. Proc. Natl. Acad. Sci. U. S. 48, 1776— 82.Chu, Yaw-en, K. G. Lark, 1976: Cell-cycle parameters of soybean (Glycine max L.) cells growing in suspension culture: Suitability of the system for genetic studies. Planta (Berl) 132, 259— 268.Collins, G. B., W. E. Yian, G. C. Phillips, 1978: Use of 4-amino-3,5. 6-trichloro- picolinic acid as an auxin source in ulant tissue cultures. Crop Sci. 18, 286— 288.Evans, D. A„ W. R. Sharp, E. F. Paddock, 1976: Variations in callus proli­feration in leaf tissue cultures of Glycine max strain T 219. Phytomorpho­logy 26, 379— 381.Fosket, D. E- J. G. Torrey, 1969: Hormonal control of cell proliferation and xylem differentiation in cultured tissues of Glycine max var. Biloxi. Plant Physiol. 44, 871— 880.Gamborg, O. L., J. P. Shyluk, 1970: The culture of plant cells with amonium salts as the sole nitrogen source. Plant Physiol. 45, 598— 600.Gosal, S. S„ Y. P. S. Baja], 1979: Establishment of callus— tissue cultures, and the induction of organogenesis in some grain-legumes. Cron Improv. 6, 154— 160.Gresshoff, P. M., S. S. Mohapatra, 1981: Legume cell and tissue culture. Proc. COSTED Symp. on Tissue Culture of Economically Important Plants, Singapore, (Ed. A. N. Rao), 11— 24.Murashige, T., F. Skoog, 1962: A  revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473— 497.Oswald, T. H., A. E. Smith, D. V. Phillips, 1977: Callus and plantlet regene­ration from cell cultures of Ladino clover and soybean. Physiol. Plant. 39, 129— 134.Petz, B., 1981: Osnovne statističke metode za nematematičare. Manualia Uni- versitatis Studiorum Zagrabiensis.Pevalek, B., 1979: Djelovanje fitohormona na rast, morfogenzu i genetsku stabilnost tkiva boba u kulturi in vitro. M. Sc. Thesis, Univ. Zagreb.Polacco, J. C., 1976: Nitrogen metabolism in soybean tissue culture. I. A s­similation of urea. Plant Physiol. 58, 350— 357.Polacco, J. C., 1976: Nitrogen metabolism in soybean tissue culture. II. Urea utilization and urease synthesis require Ni2+. Plant Physiol. 59, 827— 830.Snedecor, G. W., W. G. Cochran, 1976: Statistical Methods. The Iowa State University Press, Ames, Iowa, USA.Witham, F. H., 1968: Effect of 2,4-dichlorophenoxyacetic acid on the cytokinin requirement of soybean cotyledon and tobacco stem pith callus tissues.Plant Physiol. 43, 1455— 1457.A C T A  B O T . C R O A T . V O L . 45, 1986. 69R E N A T A  P A V L IN AS A Ž E T A KUTJECAJ 2,1-D I KINETINA NA KALUSNU KULTURU SOJERenata Pavlina(Fakultet poljoprivrednih znanosti, Zagreb)Postavljen je trofaktorijelan pokus ( 2x2x3)  u 6 ponavljanja u svrhu ispitivanja utjecaja 2,4-D (0,1 i 0,3 mgl-1) i kinetina (0,0 i 1,0 mgl-1) te eksplantatskih izvora (kotiledone, hipokotil ili vegetacijski vršci) na kvalitetu i težinski prirast kalusa soje (Glycine max /L./ Merr, linije 146/82-5). Bodovne procjene kvalitete kalusa testirane su HI2-testom (Tab. 1). Ustanovljena je različitost bodovnih procjena po harmonskim kombinacijama u odnosu na pretpostavku da razlika u kvaliteti nema. Parcijalnim testiranjem parova (Tab. 2) izdvojen je kao kvalitetniji kalus na kombinacijama auksina s kinetinom nego bez kinetina. Istosmjerna ocjena je moguća analizom vrijednosti težinskih izmjera (Tab. 3) provo­đenjem F-testa i t-testa (Tab. 4). Optimalna kombinacija za težinski pri­rast kalusa je 0,3 mgl-1 2,4-D i 1,0 mgl-1 kinetina. Nije uočena statistički značajna razlika između kultiviranih kotiledonskih ili hipokotilskih odsje- čaka ili pak vegetacijskih vršaka.Renata Pavlina, mr. sci,Fakultet poljopriveđnih znanosti Zavod za oplemenjivanje bilja, genetiku i metodiku istraživanja Simunska 25YU-4100 Zagreb (Jugoslavija)70 A C T A  B O T . C R O A T . V O L . 45, 1986.

Utjecaj 2,4-D i kinetina na kalusnu kulturu soje

Abstract

Similar works

Full text

Available Versions

Hrčak - Portal of scientific journals of Croatia