Paper 3


IBA induced new root formation




Department of Biochemistry, Indian Institute of Science, Bangalore – 560 012, India


(Revised received 23 July 1978)



Abstract – Indole Butyric Acid (IBA), an auxin, initiates roots in the hypocotyls tissue of Phaseolus vulgaris (French bean). The response in dependent on the concentration of IBA and the duration of exposure to the hormone. IBA enhances the rate of total protein synthesis in ca 30 min after exposure of the hypocotyl segments to the hormone. There is no detectable change in total or poly (A)-containing RNA synthesis in this period although significant increases are seen 2 hr after hormone pre-treatment. The early IBA – medicated increase in protein synthesis (30 min) is not sensitive to Actinomycin D but the antibiotic blocks the increase manifested 2 hr after hormone pre-treatment. Inhibition of early protein synthesis by cycloheximide depresses and delays root initiation.  Cytosol prepared from IBA – treated hypocotyls tissue stimulates protein synthesis in vitro to a greater extent than that of the control.





Adventitious root initiation in stems of dicotyledons is a phenomenon controlled and regulated by phytohormones, which bring about redifferentiation of pericyclic cells into root primordials.  In the scheme for the sequence an increase in RNA synthesis and shift from monosomes to polysomes have been proposed [1]. These changes have been detected ca 2-3 hr after auxin treatment [2]. Thus, an early increase in m RNA Synthesis is indicated, although polysomal formation need not strictly depend on mRNA availability and could be the result of ribosome activation [3-5].


In view of these results, it was felt necessary to examine the rates of some of the macromolecular events at very early periods after auxin treatment. In the present study the rates of RNA and protein synthesis have been measured within 2 hr after Indole Butyric Acid (IBA) treatment of the French bean hypocotyl tissue.



Some of the basic parameters influencing root formation in the present system were first determined. The results presented in Table 1 indicate that the number of roots produced by the IBA is proportional to the concentration up to 8-10    m g/ml.  Concentrations up to 30 mg/ml is used, higher concentrations though not inhibitory, do not significantly increase root formation. It is possible to induce root formation not only with 2 cm length segments (placed either upright or upside down), but also in segments, which are vertically split into four strips (data not given).


Table 1. Effect of IBA concentration and duration of treatment on root production in the hypocotyls segment of Phaseolus vulgaris


IBA Concentration           No. of roots           Duration of IBA     No. of roots

   (m g /ml)                       formed / segment   treatment (min)       (formed /segment

                                       (Average of 10                                      Average of

                                        segments)                                             segments)



0                              1.2 ± 0.6                        2                  19.1  ± 1.0

          4                            45.2 ± 1.3                        4                  42.7 ± 2.5

          6                            58.3 ± 1.8                        6                  69.7 ± 6.5

          8                            71.1 ± 1.7                        8                  76.6 ± 4.0

          10                          82.8 ± 1.4                        10                77.9 ± 1.7

          12                          77.6 ±  1.4                       12                73.8 ± 2.6

          20                          75.8 ± 1.6                        20                76.6 ± 1.5

          30                          77.9 ± 1.3                        30                73.6 ± 3.3                                   


Hypocotyl segments (10 segments of 8 cm length each) were placed erect in flask containing 100 ml of half strength Hoagland’s medium (basal medium). IBA concentration on the number of roots formed was examined when the tissues were exposed to the hormone for 30 min and then placed in fresh basal medium. The effect of the duration of the hormone treatment was examined at an IBA concentration of 10 mg/me. The number of roots formed 72 hr after transfer of the treated hypocotyls to fresh medium was counted. * Mean ± Standard error


Table 2. Effect of cycloheximide and chloramphenicol treatment on root initiation by IBA in the hypocotyls tissue of Phaseolus vulgaris


Pre – treatment                                               No of roots formed / segment

                                                                      (Average of 10 segments)*

                                        72 hr                      96 hrs                     120 hrs


Basal medium (BM)         Nil                         Nil                         Nil

IBA                                  77.6 ± 1.3              79.8 ±1.4               80.1 ± 5.3

IBA + cycloheximide          Nil                      Nil                         Nil

IBA +chloramphenicol     72.2 ± 2.2              78.6 ± 1.9              74.1 ± 2.6






The hypocotyls segments of 8cm length were treated with IBA (30ug/ml).  CHI (10ug/ml) or chloramphenicol (40ug/ml) was added to some flasks.  The segments were exposed to IBA plus or minus additives for 30 min, washed thoroughly and then transferred to fresh basal medium.  The number of roots formed was counted at different time intervals.  * mean+/- plus/Minus standard error.


Several cells of the pericycle, situated near and between small phloem patches and lying opposite the split exarch xylem elements, start dividing within 24 hr of auxin treatment. Many of these cells also have large nuclei with conspicuous nucleoli. During the next 24 hr the divided cells of this region appear grouped.  At the end of 72 hr after auxin treatment, the clusters of divided cells are distinctly organized as root initials in four rows reflecting the Tetrarch nature of the vasculature below. An invariant 4-row pattern of adventitious root production is maintained from the base up to the cotyledonary node.


In order to understand the biochemical processes which are activated on exposure of the hypocotyls tissue to IBA, the effects of cycloheximide and chloramphenicol on root initiation were examined. The results presented in Table 2 indicate that if cycloheximide is present during the fit 30 min of IBA treatment, the initiation of root initials is considerably delayed and the number of roots ultimately formed is also decreased. Under similar conditions, chloramphenicol has no effect on root initiation induced by the hormone. The results indicate the possibility that the hormone that the hormone may have an early effect on cytoplasmic protein synthesis, which may determine the subsequent sequence of events.


Thus, it was of interest to examine the effect of IBA on early protein synthesis. It was also pertinent to examine how long the inhibitory effect of cycloheximide on protein synthesis persisted. For this purpose, the hypocotyls tissue was treated with IBA or IBA + cycloheximide on protein synthesis was estimated immediately as well as at different time intervals after transfer to the fresh medium using [14C]- chlorella hydrolysate. The results presented in Table 3 indicate that exposure of the hypocotyls tissue to IBA for 30 min results in a significant increase in the rate of total protein synthesis. The inhibitory effect of cycloheximide is significant up to a period of 30 min after transfer to the fresh medium. However, 2 hr after the transfer, there is no significant inhibition of protein synthesis.


The effect of IBA on early RNA synthesis was next examined. For this purpose, the hypocotyls tissue was exposed to IBA for 30 min, thoroughly washed and transferred to the fresh basal medium. RNA syntheses were measured immediately, as well as 2 hr after transfer to the fresh medium using [32P] as well [3H]-uridine]. Total RNA as well as poly (A) – containing RNA syntheses was measured in these experiments. IBA–pre-treatment had no effect on total RNA or poly (A) - containing RNA synthesis when measured immediately after transfer to the fresh basal medium. However, after 2 hr there is a striking increase in the rates of total RNA as well as poly (A)- containing RNA synthesis (Table 4 and 5).



Table 3. Rate of protein synthesis after exposure of the hypocotyls tissue of Phaseolus vulgaris to IBA and cycloheximide

                        Immediately after transfer           30 min after transfer        2 hr after transfer

                        To the fresh basal medium        to the fresh basal medium   to the fresh basal


Pre-treatment    Cpm / mg cpm / mg     (B/A)        cpm/ mg        cpm/ mg     (B/A) cpm/ mg (B/A) cpm/ mg

                        Amino acid                                amino acid protein         amino acid          protein   (B/A)

                        Pool (A) (B)                                pool (A)          (B)       pool (A)            (B)


Basal medium                26.8      39.38                1.46      24.8      36.95    1.49      28.0      42.84    1.53

Basal medium + IBA      18.0      59.2                  3.28      22.6      66.30    2.96      21.6      59.4      2.75

Basal medium + IBA     

+ Cycloheximide            20.2      23.1                  1.15      23.0      38.3      1.66      17.0      52.0      3.05                                                                 


Hypocotyl tissue (1 g, 4 cm length) was placed in 5 ml of basal medium with or without (BA (30 mg/ml)/. To some flasks containing the IBA medium cycloheximide (10 m g/ ml) was added. The segments were exposed for 30 min washed thoroughly and transferred to a fresh basal medium. Protein synthesis was followed at different time intervals after transfer to the fresh transfer to the fresh basal medium using [14C] chlorella hydrolysate (10    Ci/5ml). The labeling period was 30 min.







Table 4: Effect of IBA on RNA synthesis in the hypocotyls tissue of Phaseolus vulgaris;


Immediately after transfer                              2hrs after transfer

          To fresh basal medium                                  to fresh basal medium


          Ntds pool     Total     Poly (A)              Ntds Pool     Total        Poly (A)

                              RNA     RNA                                       RNA            RNA

(Cpm/A260 unit)                                  (Cpm/A260 unit)


Basal 22500    2430           678               18500           2510             692


Basal  20600           2330             626               19600           3520             1200



The experimental details are ass given in Table 3.  RNA synthesis was followed at different time intervals after transfer to the fresh basal medium using 32P (200uci/10ml/10gm tissue).  The labeling period was 30 min.        


 In view of these results, the Actinomycin-D sensitivity of IBA-mediated early increase in the rate of protein synthesis was assessed.  The results (Table 6) indicate that Actinomycin D treatment of the hypocotyl tissue along with IBA does not abolish the increased rate of protein synthesis detected immediately after transfer to the fresh basal medium.  However, Actinomycin D prevents the increased rate of protein synthesis after 2hrs after transfer to fresh basal medium.  In all these experiments, striking changes in the sp.radioactivity of free amino acid pool were not detected.  In separate experiments, Actinomycin D was found to inhibit RNA synthesis by over 90% at both time periods examined.


Finally an attempt was made to determine whether the enhanced rate of protein synthesis detected in vivo in hypocotyls treated with IBA for 30 min could also be demonstrated in vitro.  For this purpose ribosomes and post-ribosomal supernatant (S-150) were prepared from IBA-treated and control hypocotyls.  Protein synthesis was measured in vitro using the ribosomes and S150 fractions as described in the experimental section.  A linear reaction rate could be demonstrated at least for 30 min.  The results (table 7) indicate that the rate of protein synthesis was maximal with the combination of ribosomes and S-150 fraction prepared from the hypocotyl tissue treated with IBA for 0 min. Addition of S-150 fraction from IBA treated hypocotyl ribosomes also results in a significant stimulation of protein synthesis in vitro.



The absolute requirement of apical buds for root initiation has been suggested [6, 7].  In the present study the stem tissue obtained after excision of cotyledons, leaves, apical and maxillary buds followed by thorough washing of the hypocotyls have been found not to initiate any root in the basal medium.

Table 5:  Effect of IBA on RNA synthesis in the hypocotyls tissue of P. vulgaris.L


Pre-treatment         Immediately after transfer          2hr after transfer to fresh

                              To fresh basal medium                        basal medium

                              Total RNA   Poly (A) RNA   Total RNA Poly (A) RNA

                              [Cpm/A260 unit]    [Cpm/50       [cpm/A260 [cpm/50

                                                            A260 units]     A260 units]

--------------------------------------------------------------------------------------------Basal medium        143                         35                153               42

Basal medium        159                         39                309               96

Plus IBA


The experimental details are shown given in table except that the RNA synthesis was followed was using uridine [3H] 9500uci/5ml/5 g tissues)

In view of these results the Actinomycin D sensitivity of the ribosomes indicates that some soluble factors induced by-or in combination with-IBA are at least partially responsible for the early increases in protein synthesis.



Table 6. Effect Actinomycin on IBA – mediated increase in the rate of protein synthesis in the hypocotyls tissue of Phaseolus vulgaris.


Immediately after transfer                                              2 hr after transfer

                        To the fresh basal medium                                             to the fresh basal


Pre-treatment    Amino acid pool protein             (B/A)                Amino acid pool protein (B/A)

                        cpm/ mg amino   cpm/ mg                        cpm/ mg amino   cpm/ mg

                        Acids (A)          protein (B)                                acids)    (A)       protein (B)


Basal medium    -Act.D  20.8      21.3                  1.02                  26.2                  26.9      1.03

                        +Act.D 22.7      19.7                  0.87                  23.2                  24.5      1.06

Basal medium    -Act.D  19.8      34.3                  1.73                  21.4                  43.1      2.01

+ IBA               + Act.D 18.5      36.4                  1.97                  24.2                  21.3      0.88



Hypocotyl tissue 4 cm length, was treated with Actinomycin D (100 mg/10 ml/g) for 15 min before the addition of IBA addition, the tissue was washed thoroughly and placed in a fresh basal medium. Protein synthesis was followed at different time intervals after transfer to the fresh basal medium using [14CJ-chlorella hydrolysate (10mCi/5 ml). The labeling period was 30 min.



Table 7. Effect of IBA-pre-treatment of the hypocotyls tissue on protein synthesis in vitro

Treatment                        Leucine [H3H] incorporated

                                                  (cpm / A 260 unit)

          Expt I           Expt II

Control polysomes+                            10300              11100

Control S 150 +                                   29500              36300

IBA – S 150                                        21720              123800

IBA polysomes +                                12400              114500

Control S 150                                      12400              114500



The incubation conditions are given in text. The incubation period was 70 min. The results obtained in two independent experiments are given.




Morphological studies. Phaseolus vulguris L. var. Butpees atcingtesa stingless raised in sand in a dark chamber. When the hypocotyls attained a height of 20-25 cm, they were cut l5 cm below the cotyledons and washed for 30 min in running H2O. Hypocotyl segments of 4 or 8 cm length were kept in a suitable vol. of half strength Hoagland's nutrient soln. (8) with or without IBA. In all the long term expts., a 12 hr day period was maintained . The medium was changed every day: To reduce bacterial contamination, 30 mg/ml of Ampicillin (Hindustan Antibiotic Limited) was used. The number of root initiate wart counted at different periods of time.


For anatomical studies serial hand sections were made, stained with 2 a (W/V) sacrament and observations were made under the microscope.


Protein synthesis in vivo: Protein synthesis in the hypocotyls tissue was followed by placing 1 g of suitably treated and washed segment in 5 ml medium containing 10mCi of (14C)-chlorella hydrolysate. The different treatments given are described in the table. After 30 min of labeling, the tissues were thoroughly washed to remove adhering radioactivity and a tissue homogenate was prepared by grinding in NaPi buffer (0.1 M pH 7) in the cold.  HCHO4 (0.3 N in final conc.) was added to the homogenate and the ppt, was recovered by centrifugation after standing a few hr in the cold. Protein was extracted from the ppt. with NaOH; the extract neutralized and re-precipitated with TCA (10%w/v, in final concentration).  The precipitate was subsequently washed with hot TCA, EtOH-Et2O (3:1) and finally with Et2O.  Final preparation was dissolved in HCO2H and aliquots were counted on whatman No 3 filter paper discs.  Aliquots were also used after neutralization for measurement of protein content by the method of ref [9].  The sp.radioactivity of the amino acid pool was estimated in the HclO4 supernatant.  The supernatant was neutralized and aliquots were used for radio activity measurement and total amino acid was estimated using ninhydrin by the procedure of ref. [10].


Protein synthesis in vivo:  For this purpose polysomal fraction was isolated from the hypocotyls tissue.  Isolation procedures and in vitro amino acid incorporation were similar to those described if ref. [11].  Briefly, the tissue was homogenized in 5 vols. Of buffer A containing Tris-HCL, pH 7.5, 50mM; MgCl2, 5mM; KCl, 15mM; DTT, 0.6mM and sucrose, 300mM. The homogenate was centrifuged at 20,000g for 15 min and 2ml of the supernatant was layered over 3 ml of 1.5M sucrose and centrifuged at 150 000 g for 2hrs.  The pellet was suspended in buffer A.  Another batch of homogenate was centrifuged at 20 000g for 15 min and the supernatant centrifuged at 150 000g for 2 hrs.  The final supernatant was passed through a G-25 column and was used as the source of soluble enzymes (S150 fraction).  The in vitro protein synthesizing incubation mixture contained in 0.1 ml total vol: HEPES buffer, pH 7.5, 20mM; MgCl2, 5mM; KCl, 50mM; ATP, 0.4mM; GTP, 0.12mM; phosphoenolpyruvate, 5mM; pyruvate kinase, 2 enzyme unit; amino acid mixture without Leucine, 25 uM each amino acid, polyribosome, 100ug, RNA; S150 fraction, 100 ug protein; 1uCi of Leucine [3H](7Ci/mMol).  The mixture was incubated at 37^C for 30 min. 

Aliquots (20ul) were applied to filter paper discs and discs after drying were successively washed with hot and cold 5%TCA, EtOH-Et2O 92:1) and finally Et2O.  The discs were counted for radioactivity.


RNA synthesis:  It was measured using either carrier free 32P-orthophosphate (200uCi/10 ml/10g tissue) or Uridine [3H] (500 uci/5ml/5g tissue).  The labeling period was 30 min.  After thorough washing, RNA was isolated from the tissue homogenate using PhOH-CHCl, extraction procedures [12].  Poly (A)-containing RNA was isolated from a known amount of total RNA using poly (U)-Sepharose columns [13].  For measurement of nucleotide pool sp.radioactivity in experiments using 32P, the tissue homogenate was treated with HClO4 (0.3N final conc.).  After standing for a few hr in the cold, it was centrifuged and the free nucleotides in the supernatant were adsorbed using activated charcoal.  The charcoal pellet was washed repeatedly with H2O and finally the nucleotides were eluted using 50% EtOH, 0.3N NH4 OH [14].  Aliquots of the eluate were used for radioactivity and A260 measurements.  Radioactivity of the filter paper discs was measured in a scintillation counter with 10 ml of toluene containing 0.5% PPO (w/v) with an efficiency of 6% for 3H and 35% for 14C.

Acknowledgement:  GRK thanks the University Grants Commission, New Delhi for financial help.



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