Some Varieties of Pisum sativum. substance in the light. the heredity of the pea, can reach a length of 4 m. short-stemmed varieties.

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1 On the Differences in Longitudinal Growth Some Varieties Pisum sativum by Iz. de Haan and Chr.J. Gorteṛ CONTENTS 1) Introduction 2) Analysis the longitudinal growth a) Number and the internodes b) Number and the cells 3) The osmotic conditions 4) Investigation the growth substance a) Production growth substance in the dark b) Production growth substance in the light c) The inactivation growth substance d) The demonstration oxidizing enzymes j) Summary 6) Literature 1) INTRODUCTION During his researches on the heredity the pea, H. d e H a a n in 1927 has obtained a variety, which showed an extraordinary longitudinal growth. This variety, called slender pea ( rankerwt see H. d e H a a n 1927), can reach a 4 m. It was found in the F2 two short-stemmed varieties. After further genetical examination, the slender pea appeared to be recessive in two genes, both parents being dominant for one them. The genes which cause the strong growth in are La and Lb; the slender pea is recessive in both and is therefore denoted by the formula la la lb lb. The short-stemmed varieties, from which the slender pea originated, are white with little wax ( wit met weinig was ), formula La La lb lb, and white raisin ( witrozijn ), formula la la Lb Lb.

2 435 The slender pea has an average about 335 cm, the white raisin about 90 cm and the white with little wax about 70 cm. The presence one dominant gene therefore causes an inhibition growth from 335 cm till about 90 cm. H. de Haan published in 1931 a larger paper about his genetical researches on Pisum sativum. In Chapter III the factors for are dealt with in detail and on page 416 a description the slender pea is to be found. It appears that the great the slender pea is due to the greater number internodes as well as to their greater. The slender pea forms on an average 35 internodes, while the a single internode can be as much as 17 cm; white raisin has about 24 internodes, the longest which can be 7.5 cm; white with little wax forms about 27 internodes, the longest is also 7.5 cm. H. d e Haan stated that the average pea is greater the epidermis cells the slender than that the short-stemmed varieties. It is therefore probable that the strong growth in is partly to a greater cells also. Apart from longitudinal growth, the slender pea due differs in many more characteristics from the short-stemmed peas. This makes it clear that the action both genes La and Lb must be a very complicated one, as presence one them can alter the type the plant in many respects. This will be made still more apparent by this research, which is meant as an analysis the growth in the slender pea and one its parents. In the first place the growth in the type slender pea (la la lb lb) was examined and compared with one the parents, viz. white raisin, la la Lb Lb. The differences observed in the physiology the longitudinal growth these two varieties is accepted to be founded, as far as genetically known, on the action the gene Lb. 2) ANALYSIS OF THE LONGITUDINAL GROWTH a) Number and the internodes Measurements have been made by H. de Haan (1931) about the relation slender pea and both parents the short-stemmed type. From his data it appears that the great difference in is due in the first place to a greater number mternodes in slender pea and secondly to their greater average. One us has repeated his measurements with the same result; a few the data are recorded in the following tables.

3 436 Table I; slender pea ; and number internodes. No. total number internodes average internodes i) J Î cm cm 2) J-4-J J J-IO.J J-16.J-12.J j-i J-10-8.J-8.J cm i cm 3) j- i-io.j-ii.j-10-i j j cm cm Table II; white with little wax ; and number internodes. No. total number mternodes average mternodes i) r Î-Î-Î-Î Î-J-Î-Î-6-Î r cm cm 2) I-I cm cm 3) cm cm

_ 437 Table III; white raisin ; and number internodes. No. total number internodes average internodes i) i. 5-1.5-I-I.j-2.5-2.5-3-3.5- Î.J-5-Î-6-5-6.S-7-6.J-7-6.5- Î-Î.5-5.5-4- 1 98.0 cm 23 4.

4 _ 437 Table III; white raisin ; and number internodes. No. total number internodes average internodes i) i I-I.j Î.J-5-Î S-7-6.J Î-Î cm cm 2) j-4-j.j-7-j.j-j. 7-7-Î-7-6.S-6.J-6-6-J M xi 5.0 cm cm 3) j Z j I-5 T05.0 cm cm When comparing the data in the different tables, we are struck by the fact that the slender pea has a greater number internodes, which are on an average greater. b) Number and the cells. Next we can put the question whether the greater the internodes is due to number or size the cells or to both. H. d e H a a n has established that epidermis cells slender pea are longer than those the short-stemmed varieties. It is necessary, however, to know the exact number the cells, formed by the different varieties, and their average. We have done such measurements in the following way. same In order that the three types should grow under exactly the circumstances, they were sown in one and the same bowl; germination took place in a temperate hothouse. About three weeks after sowing the first three internodes were full-grown and these the exact number cells was counted and their average measured. From the cotyledons upward the stems were cut into pieces 4.5 mm; a radial section the number cortex cells was counted in longitudinal direction. The average the cells was calculated by dividing the the piece by the number cells. This was done from below to above for the first three internodes. It might be noted that on transverse section all three varieties showed an equal number cortex cells. In the following tables the data are given for the second and third internode only; the first internode, having

5 438 grown partly beneath the surface the soil and etiolated, did not give reliable data. therefore being Table IV; slender pea. number average internode cells cells No. i 2d internode 3 6 mm /* 3d S5 mm P No. 2 2d i*. 3 d 91 mm n No. 3 2d 40 mm p 3 d 6 4 mm Table V; white raisin internode number cells average cells No. i 2d internode 9 mm /x 3d» 25 mm /X No. 2 2d 12 mm /X 3d 27 mm /X No. 3 2d 8.5 mm /X No. 3d 15 mm i6i 93 /X 4 2d 9 mm /X 3d 18 mm /X Table VI; white with little wax internode number cells average cells No. i id internode 8 mm p 3d» \y mm 113 IJO p No. 2 2d y> 10 mm /1 3d» i j mm p No. 3 2d» 8.j mm 81 M O V» "S 3d» 13.j mm s No. 4 2d 4 mm f» 3d» 8 mm 123 p

439 In comparing these tables we see immediately that the great the internode the slender pea is caused by greater number cells as well as by their greater, when compared with the short-stemmed

6 439 In comparing these tables we see immediately that the great the internode the slender pea is caused by greater number cells as well as by their greater, when compared with the short-stemmed varieties. Differences between white raisin and white with little wax are not worth mentioning. As far as anatomical examination goes, the difference in between adult plants slender pea and the short types are therefore due to: i) more internodes, 2) more cells, 3) longer cells. 3) THE OSMOTIC CONDITIONS. The reason why the slender pea forms more internodes, more and longer cells per internode than short-stemmed varieties, is probably to be found in the action phytohormones. Before investigating the production auxins, we have to consider first the osmotic conditions in the slender pea as compared with the short types. Greater cell elongation, indeed, may also be due to greater water intake, caused by higher osmotic concentration the cellsap. It has to be made out which the two is the reason the stronger cell elongation in the slender pea. Of special interest for this question is the tissue which is about stretching or in which stretching has come to an end, but which is not yet fixed by growth. The top the young stem the pea is hooked; stretching takes place just below the curve. As this tissue is st and has small cells, It is Impossible to osmotic data get from observations on one cell. The sources error, caused by preparation, would be too great. For us, moreover, it was only interest to see if there are important differences, if any. Seeds the type white with little wax running short, we could only compare the tissue slender pea with white raisin. We worked in the following way; seeds both types were sown if} same bowl and germinated under exactly identical conditions in a temperate hothouse. When the seedlings were a few days old, cylinders a lenght 2 mm were cut out the stretching zone the stems. These cylinders were put oil and their was determined; the paraffine in paraffine oil was then removed with a piece filtering paper and replaced by water. After some time (about 13 minutes) the increase in was measured. Then, with a sharp razorblade, the cylinders were cut into four pieces, which were transferred into saccharose solutions ascending concentration. The concentrations are given in grammolecules per Liter. There were, therefore, four solutions, one piece in each them. The solution, in which the cells show a beginning plasmolysis, was looked for. The the pieces,

7 plasmolysed plasmolysed 440 in which this was the case, was measured. The difference in between the cylinders in water and the pieces after plasmolysis gives the contraction, due to disappearance turgor. Table VII; slender pea. number the plant I the tissue in paraffine oil the tissue in water the tissue after plasmolysis ) concentration the limit-plasmolyticum g 0-35 g 0-35 g g differ, in water plasmolysed elongation differ, in = water in % plasmolysed (= 100) 30.7 % 10 % 7-8 % 14-2 % Table VIII; white raisin. number the plant i the tissue in paraffine oil P the tissue in water the tissue after plasmolysis l concentration the limit-plasmolyticum g g 0.40 g g differ, in water plasmolysed elongation differ, in = water in % plasmolysed (= too) 13-7 % 3-5 % 11.2 % 4-9 % 4) The cylinders No. 2 have been in the water for a longer time than the other ones, viz. 30 min. The slender pea, after plasmolysis, did not regain the initial measurements, is therefore plastically stretched. The elongation was taken to be the difference between in water and in paraffine oil, as the maximum cell stretching in a certain osmotic concentration must be considered.

441 The elongation the cylinders after transport from paraffine oil into water is dependent on the amount water, present in the tissue on the moment w 4 hen it was cut f.

8 441 The elongation the cylinders after transport from paraffine oil into water is dependent on the amount water, present in the tissue on the moment w 4 hen it was cut f. It is, therefore, more or less arbitrary. If the tissue is about saturated with water, the elongation will be slight and the contraction in the plasmolyticum great. Allthough the number our data is inadequate to determine quantitatively the exact relation the osmotic magnitudes, yet the differences, recorded in the tables, demonstrate in a sufficient way the greater elongation the cells the slender pea. There is considerable variability in the data, which can be partly explained by the method research; it is, indeed, very difficult to cut cylinders from the stretching zone only; they ought to be straight, therefore must be taken from below the hooked tip, where sometimes stretching is partly fixed already by growth. As is to be seen from tables 7 and 8, beginning plaslmolysis appears in both types in about the same saccharose concentration. So in relaxed condition osmotic concentrations are equal. The elongation which the tissue slender pea undergoes after transport into water, is greater than for white raisin ; consequently, the cells slender pea show a stronger stretching in the same osmotic concentration the cellsap, as compared with white raisin. This must be due to a greater extensibility the cell walls and not to a higher concentration the cellsap. 4) INVESTIGATION OF THE GROWTH SUBSTANCE. The fact that the extensibility the cell walls the,,slender pea is greater than white raisin, makes it very probable that the stronger stretching its cells isi caused by a greater production growth substance (see H e y n, 1933). In order to settle this we have investigated the production growth substance young terminal buds. After two hours soaking, the seeds were set out in bowls with sawdust. After 5 days they had reached a 7.1 cm (slender pea) and 3-4 cm (white raisin) (average 10 plants). The terminal buds were cut f and put between two glassplates with wet filtering paper in a moist Petri disc,according to the method used by van Overbeek (1933, see his fig. 10). Blocks agar the usual dimensions (2 X 2 X 0.9 mm) were stuck to the stems. The auxin which diffuses into these blocks can be determined by placing them on one side a decapitated

9 442 coleoptile Avena. The degree curvature is proportional to the concentration the auxin in the block (see Went, 1927, and der van Wey, 1932). Experiments made were in a dark room (orange light, Scott filters O. G. 2) with and constant temperature humidity. Material and conditions were the same as are usual for Avena tests. According to what van Overbee k (1933) found for Raphanus sativus, it appeared that in Pisum sativum the formation auxin is also a phenomenon photosynthesis. Therefore it is necessary to grow the plants in the light; the cutting f the terminal buds and the diffusion the growth substance took place in the dark room. Fig. 1.

were 0,9 mm thick. Cylinders white raisin inactivate a. 443 a) Production growth substance in the dark. "When growing the plants in the dark room, growth is noticabiy ageotropical.

10 were 0,9 mm thick. Cylinders white raisin inactivate a. 443 a) Production growth substance in the dark. "When growing the plants in the dark room, growth is noticabiy ageotropical. Only when the seed was very new this ageotropical growth was not to be seen. Plants from two years old seed, grown in darkness, are very weak and grow in all directions. Hardly any or no growth substance is produced in this case. A terminal bud an etiolated plant gave a quantity auxin according to a curvature Avena b) Production growth substance in the light. It appeared that the quantity growth substance which can be got out the terminal buds, is dependent on the production time, that is the time during which the buds are on the agar plates. This relation between production and time is recorded in figure 1; on the abscissa are put down the numbers hours during which the terminal buds were in contact with the agar, on the ordinate the curvatures the Avena in degrees. Each point the curve means an average, got by two or three experiments; each experiment included 12 plants. It appears that in the beginning white raisin produces more auxin, but that in this type a greater quantity is inactivated. The consequence is that after three hours an equal amount growth substance is found in the agar plates; after that, slender pea has got more it than,,white raisin. It can be said that,,white raisin precedes the,,slender pea : production starts earlier and is true for the inactivation. greater and the same holds c) The inactivation growth substance. Cylinders 2 mm were cut out the stems a few cm below the terminal bud and were placed on agar plates, which contained auxin a. The plates measured 8X6 mm and larger quantity growth substance than those slender pea. The concentration auxin at the beginning and after the inactivation are to be seen in table 9. It is probable that the inactivation must be due to oxidizing enzymes. d) The demonstration oxidizing enzymes. It is obvious that the destruction growth substance has to be ascribed to the action oxidizing enzymes; it is, indeed,

11 O.J cc cc Table IX. date cone, auxin in curvature number cylinders per agar plate hours auxin eft by slender pea white raisin Aug (3 mm ) Aug (2 mm) I Nov (2 mm) X Nov (2 mm) I Nov. Dec (2 mm) I (2 mm) I ro.i 6.1 a well know fact that growth substance loses its in power presence This an oxidizing agent. opinion was supported by the paper van Overbeek (1935) who demonstrated that there is a connection between the abnormal longitudinal growth a variety and corn its amount catalase. For this reason we have tried to determine the quantity catalase in the terminal buds slender pea and white raisin. Sixteen buds both varieties were cut f and rubbed down with 10 cc water in a mortar; the extracts were filtered. One cc this extract was put into the right tube above the cock a Kluyver s fermentation apparatus (K 1 u y v e r, 1914) and there it was rapidly but thoroughly mixed with 1 cc a 3 % solution * H0O0. By opening the cock, 1 cc the mixture was then sucked up into the apparatus; this x cc mixture therefore contains jo% the original extract and 30% the H2O2 solution. The quantity gaseous oxygen, put free by the enzyme, was measured on the calibrated tube the apparatus. The quantity oxygen, remaining in the solution, was neglected. We made two determinations, one with 1 cc, the second with o.j cc extract and got the following results: 1 cc mixture extract slender pea and 3 % H2O2 gives i cc mixture extract white raisin and 3 % H2O2 gives' 0.9 cc O cc mixture extract slender pea and 3% Ff 2 02 gives 0.2 cc O a. 0,5 cc mixture extract white raisain and 3% H gives

12 445 This method is rather coarse but sufficient to demonstrate a probably large difference in quantities catalase. It must be kept in mind that the tissue extract should not be too concentrated, as H2O2 has to be present in overdose. The difference in amount catalase can also be demonstrated by means a colour reaction with guajac tincture; here too the extracts should not be too concentrated, as else the difference is not manifest. We may conclude from these experiments that the tissue extract white raisin contains more catalase than that slender pea. It is evident now that out the cut cells the tips slender pea less catalase will diffuse into the agarblodks than out the white raisin. The stronger inactivation by white raisin, compared with slender pea, is therefore comprehensible. 5) SUMMARY. An anatomical investigation was made about the difference in in two varieties Pisum slender pea ( rankerwt ) sativum,, and white raisin (witrozijn ). The greater slender pea is due to more internodes and more and longer cells. Production growth substance slender pea as well as white raisin depends on the time during which their tips were on the agar plates. Production auxin, put down in a graph, shöws for both types an optimum curve as far as time is considered. The growth substance which has diffused into the agar, is inactivated by oxidizing enzymes. In slender pea destruction is less effective than in white raisin. After hours there is about twice as much auxin present in slender pea than in white raisin. The cells slender pea are more extensible in the stretching zone than those white raisin. This be can explained by a stronger production auxin. Morever, in slender pea the growth substance is present in lower zones the stem, whose stretching zone is longer than that white raisin. This makes it probable that in slender pea the inactivation also takes place in lower parts the stem or at least takes the upperhand there. Osmotic values the cells in the stretching zone are the same for both varieties. Tips white raisin contain more catalase than those slender pea ; this explains the more effective destruction the auxin in,white raisin.

13 ad 446 If we consider the relative data for cells, number cells, extensibility the cell walls and the amount catalase, it appears that they are all the same order magnitude, as be can seen in table X. The relative data for s internodes are course greater, as the an internode is determined by X number cells. Table X slender pea white raisin ad internode 3.8 i >y j) )j 3.8 i number cells in id internode i.6 i >> )>!> )> )> 1.2 i cells in id internode 2.3 i >) )> >> 3d?» 1.1 i extensibility the cell walls 1.8 i amount catalase; ist determination 1.8 i M i It may be concluded that the cell stretching slender pea and white raisin is finally determined by their amount oxidizing enzymes. This research was started in the Botanical Laboratory the State University Groningen, Netherlands, and was continued in the Botanical Institute the State University Ghent, Belgium. We wish to express our thanks to the director this Institute, Dr. G. L. Funke, for his interest and for the translation the text. 7) LITERATURE. H a a n, H. d e, 1927;,,Length-faccors in Pisum ; Genetica 9 p H a a n, H. d e, 1931;..Contributions to the genetics Pisum ; Genetica 12 p H e y n, A. N. J., 1931; Der Mechanismus der Zellstreckung ; Rec. des. trav. bot. neerl. 28 p W Kluyver, A. J., 1914; Biochemische suikerbepalingen ; thesis, Delft, Overbeek, J. van, 1933; Wuchsstf, Lichtwachstumreaktion und Phototropismus bei Raphanus ; Rec. des trav. bot. n erl. 30 p Overbeek, J. van, 1935; The growth hormone and the dwarf type in growth in corn ; Proc. Nat. Ac. Sc. vol. 21, No. 5. Went, F. W., 1927; Wuchsstf und Wachstum ; Rec. des trav. bot. neerl p. Der Mechanismus des Wuchsstftranspor- e y, H. G. van der, 1932; tes ; Rec. des trav. bot. nierl. 29 p. 379.

The Journal of General Physiology

The Journal of General Physiology GROWTH SUBSTANCE CURVATURES OF AVENA IN LIGHT AND DARK Bx J. VAN OVERBEEK (From the William G. Kerckhoff Laboratories of the Biological Sciences, California Institute of Technology, Pasadena) (Accepted