Kerstin Uvnas-Moberg OF A SUBSTANCE WITH OXYTOCIN ACTIVITY IN ORDER TO STIMULATE PLANT GROWTH The present invention relates to the use of at least one compound in order to stimu- late plant growth. It also relates to the use of a composition in order to stimulate plant growth. It also relates to a composition comprising such a compound.

Background of the invention In most cases, the preliminary stage to a plant is a seed. The seed is a resting struc- ture. The process of germination is the absorption of water by the seed, the reactivation of metabolism and the initiation of growth. The seed contains an em- bryo; one end of this embryo, the radicle will form the root of the plant ; the other end, the plumule, will form the stem and leaves. The embryo also has cotyledons or seed leaves (one in monocots, two in dicots, and many in gymnosperms) which may be small and occupy only a small part of the seed, as in monocots, or may be large enough almost completely to fill the seed, as in beans and many other dicots.

In the germination of a monocot seedling, the radicle grows downward through the split seed coat to produce the primary root, and the shoot, encased in its protective sheath, the coleoptile, grows upward. The species Agrostis tenuis, Triticum sativufn and Lactuca sativa are all monocots. For dicots, however, the plumule is not pro- tected by a coleoptile. Instead, the plumule pushes through the soil in a"crook" form, called the plumule hook. In this way, the delicate newly forming leaves of the plumule are not damaged.

Generally, it is highly desirable to stimulate plant growth but not to stimulate weed growth. Accordingly, there has been a long-felt need to fmd a cheap agent that ef- fectively stimulates plant growth but does not stimulate weed growth. Plant hor- mones are known to stimulate plant growth. Examples of plant hormones are auxins, gibberelins, cytokinins and abscisic acid. A problem with these agents is that they

are relatively expensive and they may be toxic in high concentrations. Alternatively, herbicides such as phenoxy acids may be used in order to inhibit weed growth. A problem with herbicides is above all the high toxicity thereof. As a result, many her- bicides have been prohibited by law.

The above-mentioned problems have been solved by using oxytocin as a stimulating agent for plant growth. Oxytocin is a cheap agent that is produced in the human body. Oxytocin has shown to be a more potent stimulating agent for plant growth than the plant hormones mentioned above. The mechanism of action of oxytocin is probably by improving the nutrient uptake, transport and release via roots in combi- nation with a better photosynthesis or translocation i e redistribution of inorganic and organic compounds between tissues and inclusion in seeds or other storage or- gans. In seeds, oxytocin stimulates germination but above all most likely the turn- over and translocation of organic material from the nucleus to root and shoot em- bryos. Oxytocin may be used in order to break dormancy, stimulate germination and the growth of the germ. By a faster early growth, the plant will compete better with weed. In later stages, oxytocin may influence initiation of flower primodium and increase the survival rate of florets and seeds by better inclusion of organic matter and an increased translocation of nutrient and compounds to the seeds. This may lead to more even or bigger harvests with the same quality. Oxytocin can also effect next generations causing more rapid germination and enhanced growth by influence of genomes or seed composition.

New plants could also be developed by vegetative propagation for example from bulbs, tubers, meristimatic tissues such as buds, apical meristems, cell cultures and cuttings. Cuttings can be used for grafting or planting. Unicellular algae are propa- gated by cell division.

The action of oxytocin can also be used by stimulation of cell division and in such a way enhanced growth of plants, including unicellular algae, propagated by processes mentioned above.

It should be noted that the term"plant"within the context of the present invention includes all groups of plants as is recognized by a person skilled in the art. Such groups of plants include e g gymnosperms, angiosperms, monocotyledons (mono- cots), dicotyledons (dicots) and algae.

There are different processes described for the synthetical production of oxytocin ; commercial processes are for instance described in US patents 2,938,891 and 3,076,797.

Oxytocin was one of the first peptide hormones to be isolated and sequenced. It is a nonapeptide with two cysteine residues that form a disulfide bridge between posi- tions 1 and 6 and corresponds to the formula s s-l Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 Oxytocin (SEQ ID NO: 1) There are several oxytocin derivatives, i. e. compounds with a structure similar to that of oxytocin. The inventors have preliminary indications that other oxytocin de- rivatives than oxytocin may stimulate plant growth, as well as parts of the oxytocin molecule. Such oxytocin derivatives and parts of the oxytocin molecule with the same or similar effects on plant growth as oxytocin are generally called substances with oxytocin activity. Substances with oxytocin activity also include precursors, metabolic derivatives, oxytocin agonists and analogues displaying the same proper- ties.

WO 00/18424 discloses the use of substances with oxytocin activity for the prepara- tion of a pharmaceutical composition in order to improve cell regeneration. By cell regeneration is meant the recovery of a human or animal body by a controlled and helpful generation of existing and new cells as well as cell maturation in order to replace damaged cells. WO 00/18425 discloses the use of the same substances in order to promote growth of human and animal cells. However, none of these docu-

ments describe the generation and growth of plant cells. Accordingly, it is not previ- ously known from these documents that oxytocin and oxytocin derivatives or parts of the oxytocin molecule may stimulate plant growth.

The stimulating effect of oxytocin on plant growth is shown in the Examples. In Ex- ample 1, it was shown that oxytocin increased the amount of germinated seeds and stimulated the growth of seeds for the species winter wheat (Triticum sativum) and bent (Agrostis tenuis). In Example 2, it was shown that oxytocin increased the growth of lettuce (Lactuca sativa).

Summary of the invention The present invention relates to the use of at least one substance with oxytocin ac- tivity in order to stimulate plant growth. It also relates to the use of a composition comprising at least one substance with oxytocin activity in order to stimulate plant growth. The invention also relates to a composition comprising at least one sub- stance with oxytocin activity.

Detailed description of the invention One object of the present invention is the use of at least one substance with oxytocin activity in order to stimulate plant growth.

It is preferred that the at least one substance is selected from the group consisting of the following compounds: wherein Xi is selected from the group consisting of Cys, Mpa and nothing, X2 is selected from the group consisting of Tyr, (O-methyl-Tyr), Phe, and nothing, X3 is selected from the group consisting of Ile, Val, Hoph, Phe, Cha, and nothing,

X4 is selected from the group consisting of Gln, Ser, Thr, Cit, Arg, and Daba, Xs is selected from the group consisting of Pro, and nothing, X6 is selected from the group consisting of Ile, Leu, nothing, Val, Hos, Daba, Thr, Arg, and Cit, X7 is selected from the group consisting of Gly, nothing, and Ala, X8 is selected from the group consisting of Gly, and nothing, Xg is selected from the group consisting of CH2 and S ; as well as salts thereof.

The cystein disulfide bridge is only present when Xi represents Cys or Mpa, X2 rep- resents Tyr, (O-methyl-Tyr) or Phe, and X3 represents Ile, Val, Hoph, Phe or Cha.

By"nothing"is meant that the letters respectively may have no meaning or may rep- resent a bond and that there may be a direct bond between the items (letter, atom or group) situated to the right and to the left, respectively, of the letter designating "nothing". For example, in SEQ ID NO: 2 above, when only Xi designates nothing, the resulting molecule corresponds to X2-X3-X4-Asn-Cys-X5-X6-X7-X8-NH2. When only X8 designates nothing, the X7 residue is amidated.

It is even more preferred that the at least one substance is selected from the group consisting of the following compounds: Oxytocin (SEQ ID NO: 1) Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, XS is Pro, X6 is Leu, Xy is Gly, X8 is noth- ing, and Xg is S in Claim 2 and 6 Mesotocin (SEQ ID NO: 3) Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, Xs is Pro, X6 is He, Xy is Gly, X8 is noth- ing, and Xg is S in Claim 2 and 6

Isotocin (SEQ ID NO: 4) Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Ser, Xs is Pro, X6 is Ile, X7 is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 Annetocin (SEQ ID NO: 5) Xi is Cys, X2 is Phe, X3 is Val, X4 is Arg, Xs is Pro, X6 is Thr, X7 is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 Vasotocin (SEQ ID NO: 6) Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, Xs is Pro, X6 is Arg, X7 is Gly, X8 is noth- ing, and Xg is S in Claim 2 and 6 Vasopressin (SEQ ID NO: 7) Xi is Cys, X2 is Tyr, X3 is Phe, X4 is Gln, XS is Pro, X6 is Arg, Xy is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO: 8 Xi is Cys, Xa is Tyr, X3 is Ile, X4 is Gln, Xs is Pro, X6 is Leu, X7 is Gly, X8 is Gly, and X9 is S in Claim 2 and 6

SEQ ID NO: 9 Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5-X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO: 10 Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6-X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO : 11 Xi is Cys, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7-X8 is nothing, and Xg is S in Claim 2 and 6 Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 SEQ ID NO: 12 Xi is nothing, X2 is Tyr, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, Xy is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 SEQ ID NO: 13 Xi-This nothing, X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, Xy is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 Gln-Asn-Cys-Pro-Leu-Gly-NH2 SEQ ID NO: 14

X1-X3 is nothing, X4 is Gin, Xs is Pro, X6 is Leu, Xy is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 Ile-Gln-Asn-Cys-Pro-NH2 SEQ ID NO : 15 X1-X2 is nothing, X3 is Ile, X4 is Gln, X5 is Pro, X6-X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO : 16 Xi is Cys, X2 is Tyr, X3 is Cha, X4 is Cit, X5 is Pro, X6 is Arg, X7 is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO: 17 Xi is Cys, X2 is Tyr, X3 is Val, X4 is Thr, Xs is Pro, X6 is Leu, Xy is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO : 18 Xi is Cys, X2 is Tyr, X3 is Hoph, X4 is Thr, Xs is Pro, X6 is Val, X7 is Gly, X8 is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO : 19 Xi is Cys, X2 is Tyr, X3 is Phe, X4 is Cit, XS is Pro, X6 is Leu, Xy is Gly, X8 is noth- ing, and X9 is S in Claim 2 and 6

SEQ ID NO: 20 Xi is Cys, X2 is Tyr, X3 is Cha, X4 is Arg, XS is Pro, X6 is Hos, Xy is Ala, X8 is nothing, and X9 is S in Claim 2 and 6 SEQ ID NO : 21 Xi is Cys, Xz is Tyr, X3 is Val, X4 is Daba, Xs is Pro, X6 is Daba, Xy is Ala, X8 is nothing, and X9 is S in Claim 2 and 6 SEQ ID NO : 22 XI is Cys, X2 is Tyr, X3 is Hoph, X4 is Daba, Xs is Pro, X6 is Cit, X7 is Ala, Xg is nothing, and Xg is S in Claim 2 and 6 SEQ ID NO : 23 Xi is Cys, Xz is Tyr, X3 is Phe, X4 is Arg, Xs is Pro, X6 is Val, X7 is Ala, X8 is nothing, and Xg is S in Claim 2 and 6 CH2S-H pa- (O-methyl-Tyr)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 Carbetocin (SEQ ID NO: 24) Xi is Mpa, X2 is (O-methyl-Tyr), X3 is Ile, X4 is Gln, X5 is Pro, X6 is Leu, X7 is Gly, X8 is nothing, and Xg is CH2 in Claim 2 and 6, wherein Mpa stands for p-mercaptopropionic acid ; wherein the CH2-S-group thereof is bonded to the cystein portion via a thioether bond i position 6 giving the structure for SEQ ID NO: 24 as follows:

7 CH2 S CH2CO-(O-methyl-Tyr)-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 wherein (O-methyl-Tyr) stands for O-methyltyrosine of the chemical formula: wherein Cha stands for cyclohexylalanine, Hoph stands for homophenylalanine, Cit stands for citrulline, Daba stands for diaminobutyric acid, and

Hos stands for homoserine.

Annetocin has been isolated from the earthworm, as described in Oumi T, Ukena K, Matsushima O, Ikeda T, Fujita T, Minakata H, Nomoto K, Annetocin: an oxytocin- related peptide isolated from the earthworm, Eisenia foetid, Biochem Biophys Res Commun 1994, Jan 14 ; 198 (1) : 393-399. The uterotonic activity and myometrial receptor affmity of carbetocin is described in Atke A and Vilhardt H, Acta Endocri- nologica (Copenh) 1987,115: 155-160.

Other substances with oxytocin activity could also be used, such as naturally occur- ring or artificially modified variants, analogues, and derivatives of oxytocin, mesotocin, isotocin, and annetocin. Such substances could be obtained by addition, insertion, elimination, or substitution of at least one amino acid in these hormones.

By a substance with an oxytocin like activity is also understood precursors, metabo- lites such as metabolic derivatives e. g. metabolic degradation products, agonists, or analogues of the substances mentioned herein displaying the same properties. When one or more amino acids are added to a substance with oxytocin activity, it is pre- ferred to add 1-3 amino acids to the carboxyl terminal. Metabolic derivatives or metabolic degradation products may be oxytocin like peptides e. g. with nine amino acids such as oxytocin, mesotocin, isotocin, and annetocin from which one or more amino acids has been deleted from either the carboxyl terminal end or the amino terminal end or both the carboxyl terminal and the amino terminal end, preferably 1-

j 3 ammo acids trom each terminal. it could be ascertained that these variants are analogues of oxytocin, mesotocin, isotocin or annetocin by immunological methods, e. g. RIA (radioimmunoassay), IRMA (radiometric methods), RIST (radioimmu- nosorbent test), and RAST (radioallergosorbent test). The invention also includes substances having at least 50,60,70,80 and most preferably 90% homology to oxytocin, and showing oxytocin activity.

As mentioned above, there are indications that addition of one or more amino acids to the oxytocin molecule may give a molecule that stimulates plant growth. One ex- ample of such a molecule is SEQ ID NO: 8.

As mentioned above, there are indications that subfragments of the oxytocin mole- cule may stimulate plant growth. Examples of subfragments of the oxytocin mole- cule are the following compounds: SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

There is also a possibility to create new compounds with oxytocin activity by means of computer simulation. Methods for computer simulation are known by a person skilled in the art, e. g. as described in EP 0660 210 A2. Eight new compounds have been created by means of computer simulation, namely the following peptides: SEQ ID NO : 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23.

The invention also relates to the peptides mentioned above in both D-and L-form, and racemates thereof. Especially the invention relates to the L-form. By inversion of the peptide sequence thereof, the D-form could be converted to the L-form. The effect of the D-and L-forms are the same. These and the peptides above can be pro- duced by methods known to a person skilled in the art, e. g. according to Merrifield, P. B.,"Solid Phase Synthesis", Angew. Chemie, 1985, No. 97, p. 801.

Another object of the invention is a composition comprising at least one substance with oxytocin activity in order to stimulate plant growth, in mixture or otherwise

together with at least one suitable adjuvant. It is preferred that the substance is se- lected from the group consisting of compounds with the formula SEQ ID NO: 2. It is even more preferred that the substance is selected from the group consisting of the following compounds: SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, and SEQ ID NO: 24.

General principals in administration of growth stimulating agents are to have the substances mentioned above in mixture or otherwise together with adjuvants such as water or organic solvents such as alcohol or coating agents such as gels, which may delay the delivery of a substance with oxytocin activity. Drying such agents will delay the delivery of oxytocin until water is absorbed by the coating agents. All these agents can be used as a solvent for the substance or substances with oxytocin activity. The substance is taken up by the plant via the root or the leaf and may be formulated in order to make it more or less water-soluble. The water solubility may be regulated by binding the substance to a carrier. A carrier is an organic group making the substance more or less hydrophilic. An example of a carrier making the substance more hydrophilic is an amide group. An example of a carrier making the substance less hydrophilic is a methyl group. When the substance bound to a carrier has been assimilated to the plant, the carrier will be removed enzymatically by using a naturally occurring enzyme of the plant. Substances with oxytocin activity can be used in many ways as a stimulator of dormancy breaking, germination and growth.

One example would be to speed up germination and early growth of wheat by appli- cation of a substance with oxytocin activity to wheat seeds before sowing. In this way, wheat would overcome competition by weeds. A substance with oxytocin ac- tivity can be applied to plants at one developing stage or several different stages de- pending on which effect of a substance with oxytocin activity that is desired.

As used in the present application:

the term "fresh weight" (abbreviated fw) means the weight of the plant before dry- ing; the term "dry weight" (abbreviated dw) means the weight of the plant after drying; the term"dry matter" (abbreviated dm) means the plant materia obtained after dry- ing. When the dry matter is expressed in %, this means the ratio between the dry weight and the fresh weight for a specific part of the plant.

All publications mentioned herein are hereby incorporated by reference. By the ex- pression"comprising"we understand including but not limited to. Thus, other non- mentioned substances or additives may be present.

The invention will be illuminated by the following Examples, which are only in- tended to illuminate and not restrict the invention in any way.

Examples Statistics Statistical analysis was performed by means of 2-way ANOVA followed by Bonfer- roni's test for post hoc comparison. Results are given as mean values. Differences in the number of germinated seeds, coleoptile length, root length (Example 1) and growth of lettuce (Example 2) were assessed by Student's t-test for independent means. Results were considered significant when p<0.05.

Example 1-Germination test Materials and methods In each of 10 Petri dishes with a diameter of 10 cm, 100 seeds of bent (Agrostis ten- uis) are placed together with two filter papers. To five of the Petri dishes is added 4,5 ml of a 1 llg/ml aqueous solution of oxytocin. To the other five Petri dishes is

added the same amount of tap water. All ten Petri dishes are then placed in darkness for germination during fourteen days at a temperature of 18°C.

In each of 10 Petri dishes with a diameter of 10 cm, 100 seeds of winter wheat (Triticum sativum) are placed together with two filter papers. To five of the Petri dishes is added 4,5 ml of a 1 ug/ml aqueous solution of oxytocin. To the other five Petri dishes is added the same amount of tap water. All ten Petri dishes are placed in darkness for germination during fourteen days at a temperature of 18°C.

Results Germination of seeds of winter wheat (Triticum sativum) and bent (Agrostis tenuis) was affected by oxytocin (Table 1). The treatment denoted"Untreated"refers to treatment with tap water only. As is seen from the Table, oxytocin increased the number of germinated seeds and stimulated the growth of seeds of winter wheat.

The results were similar for seeds of 4grostis tenuis. However, only the increase in root length was significant in the latter species.

Table 1. Mean number of germinated seeds, coleoptile length and root length of Triticum sativum and Agrostis tenuis after treatment of the seeds with or without oxytocin.

Species Treatment Germinated Coleoptile Root length seeds length [mm] [mm] Triticum sati- vum Untreated 89.3 0.0 2.2 Oxytocin 97.0 27.4 36. 0 P-value 0. 005 <0. 001 <0. 001 Agrostistenuis Untreated 5.4 37. 2 12.0 Oxytocin 9.8 39. 1 15.7 P-value 0. 156 0. 524 0. 025 Example 2-Growth test Materials and methods Germination The plant used was lettuce (Lactuca sativa), sort"Calgary"and non-disinfected ecological seeds were used. In the same manner as in Example 1, 150 seeds are ger- minated in oxytocin solution and 150 seeds are germinated in tap water, in both cases in darkness.

Growth phase After germination for six days, the germs are put into 1 litre test vessels with four germs per vessel. To ten vessels are added oxytocin treated germs and to ten other vessels are added germs treated with tap water, giving totally eighty plants. Each vessel contains 1 litre 300 p, M calcium sulphate solution. Other nutrients were added

on the first and third day and from day five until harvest, 21 days after transferring germs to vessels. Increasing volumes from a stock nutrient solution (24 mM potas- sium, 2.5 mM magnesium, 22.3 mM ammonium, 4 mM phosphorus, 46.9 mM ni- trate, 1 mM chlorine and trace elements) were supplied to meet the immediate nutri- ent demand of plants with a relative growth rate of 20% day 1. Three days after the transport of the germs to the vessels, 1 mg of oxytocin was added to half of the oxytocin treated germs and to half of the germs treated with tap water giving a oxy- tocin concentration of 1 Zg/ml. The growth phase experiment was performed in a climate chamber with 14 hours illumination per day at a temperature of 18°C at light and at a temperature of 14°C in darkness.

Accordingly, the eighty plants have been treated as follows: 20 plants (5 vessels each having 4 plants)-treated with tap water during germina- tion and growth (denoted-ox/-ox); 20 plants (5 vessels each having 4 plants)-treated with oxytocin solution during germination and with tap water during growth (denoted + ox/-ox); 20 plants (5 vessels each having 4 plants)-treated with tap water during germina- tion and with oxytocin solution during growth (denoted-ox/+ ox); 20 plants (5 vessels each having 4 plants)-treated with oxytocin solution during germination and growth (denoted + ox/+ ox).

Results after the growth phase Lettuce treated with oxytocin at germination (+ ox/-ox) or during the growth in nutrient solution (-ox/+ ox) showed a significant increase in growth compared with control (-ox/-ox) (Table 2). In particular, the root growth is stimulated by oxyto- cin. The content of dry matter was also increased by these treatments. Treatment of lettuce during both germination and growth in nutrient solution (+ ox/+ ox) gave no effects compared with control. The root length was even inhibited when oxytocin was available during the whole experiment period. The same was found for the content of dry matter. The results show that oxytocin stimulates growth rate, espe- cially the root growth, and increases the accumulation of organic matter in plant tis-

sues, whereas the water content decreases. The results show that different effects may be induced depending on when and how many times oxytocin is applied.

Table 2. Growth of lettuce, Lactuca sativa, treated with or without oxytocin. Treat- ment during germination/treatment during growth,-ox = without oxytocin, + ox = with oxytocin. Numbers following of the same letter are not significant at the 5% level. This means that"a"and"b"for two values differ significantly from each other at the 5% level but not with"a"and"a"or"a"and"ab". The P-value refers to the ANOVA value for the model. ox/-OX7+ ox/-ox F-ox/+ ox + ox/+ ox P-value Whole plant, fw, g 6.33b 6.59a 6.51ab 6.41ab 0.0530 Whole plant, dw, g 0.730b 0.782a 0.797a 0.721b 0.0153 Shoot, fw, g 4.436a 4.559a 4.508a 4.466a 0.3204 Shoot, dw, g 0.603b 0.641ab 0.656a 0.596b 0.0427 Shoot, dm, % 13.60b 14.06ab 14.55a 13.34b 0. 0448 Root, fw, g 1.89b 2.03a 2.00a 1.94ab 0.0279 Root, dw, g 0.126b 0.141a 0.141a 0.126b 0.0039 Root, dm, % 6.70ab 7.03a 6.92ab 6.44b 0.1329 Root length, cm 23.4a 24.4a 23.6a 21. 00b 0.0053 Shootlroot, fw 2. 35a 2.24a 2.25a 2.30a 0.1998 Shoot/root, dw 4.80a 4.59a 4.67a 4.76a 0.6165