AU683177B2 - Method for the treatment of seeds with betaines - Google Patents

Description

WO 95/35022 PCT/AU95/00357 RECEIVED 2 2 MAY 1996 1

TITLE

METHOD FOR THE TREATMENT OF SEEDS WITH BETAINES TO INCREASE STRESS TOLERANCE, SEEDLING RIGOUR ANY YIELD FIELD OF THE INVENTION THIS INVENTION relates to a method for the treatment of seeds with betaines.

PRIOR ART Under conditions of environmental stress plants accumulate a large quantity of organic solutes containing nitrogen. One group of these solutes is broadly classified as betaines. At least 12 different betaines occur in plants. A list of the major betaines is given in Table 1 of the following reference, i.e. Wyn Jones and Storey, Betaines. In The Physiology and Biochemistry of Drought Assistance on Plants. Paleg and Aspinall (eds.) Academic Press. Sydney 1981, pp171-204 Glycinebetaine is the most common of the betaines and occurs widely in plants. A number of plants from a variety of families have been identified which have accumulated proline (Aspinall and Paleg.

Proline accumulation: physiological aspects. In The Physiology and Biochemistry of Drought Resistance in Plants. Paleg and Aspinall (eds.) Academic Press. Sydney 1981, pp97-130) and glycinebetaine (see Wyn Jones and Storey reference above). As well, betaines accumulate in native Australian plants such as Melaleuca spp. (Poljakoff-Mayber et al., 1987. Aust. J. Plant Physiol. 14 341-50, Jones et al., 1987.

Phytochemistry 26 3343-3344 and Naidu et al., 1987. Aust. J. Plant Physiol. 14 669-677).

In the laboratory betaines have been shown to protect plant processes against cold, heat, drought and salinity. Betaines have been shown to protect enzymes against denaturation through stabilization of the native enzyme structure resulting from molecular crowding (Winzor et al., 1992. Archives Biochemistry Biophysics 296 102-107). Betaines are also involved with other cellular components such as with protecting membrane proteins against dehydration and conformational changes SAMENDED SHEET i: O IR AU c- r-- WO 95/35022 PCT/AU95/00357 2 (Jolivet et al., 1983. Z. Pflanzenphysiol 109 171-180).

Because betaines have been shown to provide some protection to plants in adverse environments, they have been used to treat soils and growth media, as well as being sprayed onto the leaves of plants. German Patent Specification No. 2808365 discloses an organic mineral soil improver which includes betaine. Both British Patent Specification No. 2180529 and U.S. Patent Specification No. 4818268 describe osmoprotectant particles for enhancing the growth of mushrooms. The osmoprotectant particles comprise a carrier particle having recessed attachment sites and osmoprotectant droplets having a core of a phospholipid material surrounded by a layer of an osmoprotectant material which includes at least one betaine. The droplets are attached to the recessed attachment sites of the carrier particle. The osmoprotectant particles may be added either to the compost for the mushrooms or be brought into direct contact with immature spawn prior to planting.

Japanese Patent Specification No. 01208386 discloses a fertiliser containing betaine that can be applied to paddy fields for hydroponic cultivation or may be sprayed over leaves of plants. As well, the fertiliser can be in the form of grains or pellets.

In Bodapati et al. (Bodapati et al., 1992. 32nd Annual General Meeting of the Australian Society of Plant Physiologists Abstract No. 39), the researchers reported an increase in grain yield of coldstressed buckwheat in field conditions with the foliar application of glycinebetaine. Initial experiments involved the growing of plants in glass house conditions for three weeks at 200C. Glycinebetaine or proline at 0, 15 or 50 mM was applied twice to the foliage. Three days after application the plants were exposed to 1 oC for two days. Glycinebetaine treated plants maintained higher levels of both relative water content and chlorophyll-b than control plants. Proline application provided some positive influence on chlorophyll-b content only. For the field trials 50 mM glycinebetaine was applied to the foliage resulting in a 15% increase in WO 95/35022 PCT/AU95/00357 3 grain yield.

As well as treatment of soil and foliage, seeds have been treated with a betaine prior to germination. A group of researchers treated carrot and celery seed at -1.0 MPa (approximately 0.4 M) and onion and leek seed at -1.5 MPa (approximately 0.6 M) with betaines and compared with a control treatment with seed germinating in water for 14 days at 15C. They found the percentage of glycinebetaine treated leeks, celery and onion seeds germinating was not affected by the treatment.

However, the percentage of glycinebetaine treated carrot seeds germinating was reduced. As well, germination time for all four species was reduced compared with controls (Gray et al., 1991. Seed Sci Technol. 19 581-590).

Another group working with Kosteletzkya virginica found that the application of glycinebetaine or proline did not improve germination under saline conditions. Glycinebetaine or proline was added to the germination medium at 10 mM concentration and the seeds were incubated in 12 hour alternating temperatures of 28/18 0 C in the'dark.

After four days incubation, the germination of Kosteletzkya virginica seeds in a non-saline germinating medium showed no significant effect with the addition of proline or glycinebetaine. However, in the presence of 100 mM NaCI in the germinating medium, the germination of seeds in medium also containing 10 mM glycinebetaine or proline were inhibited compared with the germination of seeds in medium without glycinebetaine or proline (Poljakoff-Mayber et al., 1994. American Journal of Botany 81 54-59).

SUMMARY OF THE INVENTION The present invention results from the surprising discovery that the application of a betaine at a defined concentration to seeds enhances seedling growth and protects the seeds against the effects of adverse conditions in a stressful environment during germination. This was hitherto unknown before and indeed the prior art teaches that the application of a betaine to a seed had no effect on seedling growth during II I_ WO 95/35022 PCT/AU95/00357 4 germination and may even be inhibitory.

Thus it is an object of the present invention to provide a method of treating the seeds so to enhance seedling growth during germination and or protect germinating seeds against adverse conditions.

In one aspect, the invention provides a method for treating a seed to enhance seedling growth and/or protect against environmental stress during germination by treating the seed with betaine prior to planting whereby the seed is immersed in a solution if the betaine and said solution has a 0.34 M or lower betaine concentration, or the seed is coated with a solid form of the betaine at a ratio of 1-10 betaine weight per seed weight.

The seed may be obtained from any seed producing plant including those from the groups gymnospermae and angiospermae. The seed may be from a monocotyledon plant such as wheat or a dicotyledon plant such as vegetable legumes, fruit trees, and ornamental plants (e.g.

flowers). The seed may be obtained from fodder legumes such as Desmanthus virgatus and Leucaena leucocephala, or Trifolium repens.

The seed may have a hard seed coat or a soft seed coat.

Many varieties of dicotyledon plants have seeds with hard seed coats, whereas only some monocotyledon plants have seeds with hard seed coats. Suitable seeds with a hard seed coat include lupins, lecurne, castor, Leucaena leucocephala and Desmanthus virgatus seeds.

Suitable seeds with a soft seed coat include peas, raddish, cauliflower and some beans. Soft coated seeds are usually permeable except when they are in a dormant state. Soft coated seeds in a dormant state are prevented from germinating by the accumulation of germinating inhibitors and require long soaking times in which to allow the inhibitors to leach from the seed. Seeds including those with hard and soft seed coats, which are not permeable to preparations of the betaine or betaine analogue may be made substantially permeable by roughening or scarifying the surface of the seed coat prior to treating the seed with the betaine. Scarification may be achieved by mechanical means, using hot

M

PCT/AU 9 5/0 03 7 RECEIVED 19 JU1996 5 or boiling water, or subjecting the seed to acid such as sulphuric acid or alkali treatment. Scarification is preferably achieved by mechanical means. A mechanical scarifier may be fitted with coarse sand paper or the like.

The seed is preferably scarified until the seed coat is no longer glossy and scars appear on the seed coat. Not all seeds require scarification and of the seeds used as examples below, namely wheat, cotton, tomato and Desmanthus, only Desmanthus may require scarification.

The term Environmental stress as used in the present specification is one or more of the following: water stress (flood and drought), excess NaC1 (salinity), temperature extremes (heat and cold), pH extremes (acidic and alkaline soils) and heavy metal toxicity.

The term "germination" includes the development.

into a plant or individual from a seed.

Where used herein the term betaine includes amino acid derivatives where the nitrogen is fully or partially methylated. Some betaines may have sulphur substituted for nitrogen. A betaine may be any molecule with one of the following formulas

(CH

3 3 -n (R)n-N-CH 2 -X (I)

(CH

3 3, (CH 2 -X a) (CH 3 -n(R)n-N -(CH 2

R

1 (CHaR) -X (a) -CH(R) -X (II)

R

2

III)

R

2

H

4> silaffyankalkeeplspoclPCT.AU95 16.7 v, 0 0 AMENDED SHEET

PEA/AU

1eCMAU 9 t) U U) b RECEIVED 1 9 JUL 1996 5/1

-X

(3:v)

(V)

(VI)

(Vaa) R R R 2 x

R

(Ia x wherein n is 0, 1lor 2 m is 1 or 2 R is chosen from the group comprising alkyl and aryl. groups R L and R 3 are independently chosen from the group comprising H, alkyl. groups, carboxcyalkyl groups and side chains of amino acids s et(0yfkoapPCt.AU95..I 22.5.96 AMENDED SHEET

~PEAAU

Pcr/Au 95 0 0 35 7 RECEIVED 1 9 JUL 1996 5/2

R

2 is chosen from the group comprising OH, halides, alkyl groups, NH 2 and electrophilic groups R' is chosen from the group comprising H and alkyl groups and X is chosen from the group comprising 0 0- and -P -o- 0 H Betaine preferably includes glycinebetaine and those betaines listed in Table 1.

The betaine concentration in solution may depend on the type of seed, whether the seed is or has been made permeable, the betaine type, and the length of time the seed is immersed in the solution. The duration of soaking is preferably for at least two hours but may not be greater than 12 hrs. Soaking the seed in the betaine solution may facilitate the entry of the betaine into the seed whereupon it may be available for use by the germinating seed. In the case of wheat, it has been demonstrated that 40-80 g betaine per kg of seed in 2 litres of water and immersed for 12 hrs prior to drying and planting in pots produced a germination rate that was more than double the germination rate of the control under 0.2 M salinity conditions. As well, there was a four fold 21.5,06 RA4 AMENDED

SHEET

.IPEA/AU

n? WO 95/35022 PCT/AU95/00357 6 increase in wheat grain yield compared with control, The solution of betaine may also contain wetting agents and/or surfactants which assist in permeating the seed.

Soaking may occur at any temperature but preferably at 25 0

C.

The concentration of 0.34 M approximately corresponds to g/L of glycinebetaine which is the highest concentration at which beneficial effects were observed. Beneficial effects were also noted with the use of lower concentrations. It is suggested that high concentrations of betaine are viscous and have lower osmotic potential and inhibit the uptake of betaine by the seed.

After treating the seed with the preparation of betaine, the seed may be dried if it is not to be used immediately, The seed is preferably dried to a stage where it can be stored in normal commercial storage conditions without loosing its ability to benefit from the treatment upon germirztion. The seed is preferably dried to a stage whereupon storing it does not rot. The seed may be dried to a stage where it contains only about 10% moisture.

After immersion or soaking the seed may be dried and may be coated with a drying agent. This may be necessary as betaine is hydroscopic. Suitable drying agents include lime, gypsum, dolomite, rock phosphate and several clay minerals (montmorillonite and vermiculite).

The betaine soaked seed may also be coated with an adhesive to ensure the retention of betaine. Suitable adhesives include methyl cellulose and gum arbica which comprises a mixture of plant extracted gums, A further coating of a drying agent may be added. These coatings may be added stepwise or a drying agent and adhesive may be added together. Other coatings may be added to facilitate the slow release of betaine, A suitable example may be a betaine-soaked seed coated in lime and methyl cellulose.

A solid form of betaine may consist of betaine in a powder.

Other additives such as fillers, adhesives and drying agents may also be WO 95/35022 PCT/AU5/00357 7 added. Preferably a seed is coated with an adhesive, then a betaine coat and then coats of a drying agent and an adhesive. Betaine may be mixed with inert fillers such as fine sand. Other additives may be included to effect slow release of the betaine. Fertiliser may be combined with betaine coated seed. With the onset of rain or irrigation, the betaine coating the seed will become available to be absorbed by the seed or roots. In the examples below, betaine is applied to the soil simulating release from the coat material. With respect to these seeds, it was estimated the amount of betaine required was approximately 1, 1, 5 and 3-5 kg per kg of seed for wheat, cotton, Desmanthus virgatus and tomato respectively.

The methods of applying the various coatings are standard techniques (Scott, 1989, Advances in Agronomy, Ed. Brady, Vol. 42, pp 44-77, Academic Press, San Diego, Irrespective of whether the betaine is applied in a solution or coating on the seed, the concentration of betaine available for use by the seed is important and is achieved by immersing the seed in betaine solution of 0,34 M or lower concentration or by coating the seed with betaine at an amount of 1-10 betaine weight/seed weight.

In a second aspect, the invention provides a seed tr'lted by the aforementioned method.

Reference may now be made to various preferred embodiments and it should be noted that the references to specific seeds, betaines and concentrations are given by way of example only.

EXPERIMENTAL

EFFECT OF BETAINE SEED TREATMENT ON GERMINATION, GROWTH AND YIELD OF WHEAT, COTTON, TOMATO AND DESMANTHUS GROWN IN SALINE OR WATER STRESSED

CONDITIONS

WHEAT

WO 95/35022 PCT/AU95/00357 8 1.1 Seed soaking time The viability of soaked and re-dried seed depends on the length of soaking. An experiment was conducted to find an optimum time required to soak seed in water without any adverse effects on germination and vigour.

Ten gram lots of wheat (Triticum aestivum cv. Hartog) seed was soaked in small beakers with 20 ml of de-ionised water at 20"C for 0, 2, 4, 8, and 12 hrs with four replications. At the end of each soaking period, seed was weighed and re-dried for 4 hrs at 45 0 C. Germination and vigour tests were done in petri dishes with 11 cm diameter. Ten seeds were placed in each petri dish on a filter paper with 12 ml of deionised water. The experiment was conducted in an incubator with 22/18"C day/night temperature with 12 hrs of photo period with a photon flux density of 75 pEm 2 At the end of 3 days of germination, shoot and root lengths were measured.

Wheat germination and vigour which is measured as shoot and root lengths showed no difference in relation to even 12 hrs of soaking (Table It was also clear that imbibition was complete by 8 hrs soaking. These results suggests that soaking of seed with a betaine solution will have no adverse effect up to 12 hrs of soaking and re-drying.

1.2 Effect of betaine on germination of salinised wheat in petri dishes Seed was soaked in 5, 10, 20, 30 or 40 g/L solution of betaine for 12 hrs. The weight of the betaine solution added was twice the weight of the wheat seed. Then the seed was rapidly dried in a fan forced oven at 45 0 C for 4 hrs. This seed with untreated control seed was used in petri dish salinity experiment. The conditions for this experiment were same as in 1.1 except that the germinating medium, contained 3 levels of salt solutions, 0, 0.15, and 0.2 M. Seedling height and root lengths were measured 10 days after the start of the imbibition.

Betaine showed no significant effect on the shoot or root WO 95/35022 PCT/AU95/00357 9 length of wheat when there was no salt in the germination medium.

However, at both 0.15 and 0.20 M salinity levels, seedling vigour was significantly reduced in the absence of betaine seed treatment. On the other hand, seed imbibition of betaine increased shoot length at both the salinities tested. Betaine effect was maximum at 20 g/L and this effect starts reducing with higher betaine levels.

1.3 Effect of betaine imbibition on the germination of wheat in salinised pots Plastic pots fitted with plastic lining bags to cover the drainage holes were filled with 1.8 kg Samford loam. Each pot was watered with 450 ml of 0, 0.15 or 0.2 M NaCI solution with 4 replications.

Betaine levels of 20, 40, and 60 g/L were imbibed in a similar manner to the one described in section 1.2. Ten seeds were planted per pot and maintained in a growth cabinet with 22/18Cday/night, 400 pEm" 2 Germination and emergence counts were taken every day for the first days. Then plants were fertilised with Aquasol at 8 g/L as per the manufacturers recommendation. The pots were weighed every alternative day and water used up is added to make up 450 ml/pot. Plants were maintained until the maturity under these conditions. The results are shown in FIGS. 1-3.

Wheat showed a germination of 98% in non-saline soil (FIG.

This was reduced significantly by 0.15 and 0.2 M salinity to 52 and Betaine imbibition showed no difference on germination when there was no salinity. All betaine levels significantly increased germination at both at 0.15 and 0.2 M salt levels (FIG.2). Maximum benefit was achieved with betaine at 40 g/L and 0.15 M salinity. Further increase in betaine level showed a reducing trend in germination. However, at 0.2 M salinity, betaine was showing an increasing trend even at the highest betaine concentration of 60 g/L. This indicates that a specific soil salinity requires a specific dose range of betaine for useful effect on germination.

The data from the same experiment shows that salinity significantly reduces grain yield (FIG. At 0.15 M salinity grain yield WO 95/35022 PCI/AU95/00357 was significantly increased by about 30% with the use of betaine compared to the use of non-betaine imbibed seed. This effect was very dramatic at 0.2 M salinity level where, the yield increase was about 400% more than with the use of non-betaine imbibed seed. At both the salinities the use of any higher levels of betaine was not beneficial.

1.4 Effect of betaine on wheat in saline medium in petri dishes This experiment is similar to the one described in the section 1.2 except that betaine at 0, 1, 2, 4, 8, 16 and 20 g/L was added in all combinations to the solutions containing 0, 0.1, 0.15 and 0.20 M NaCI with 4 replications. Seedling height and root lengths were measured 10 days after the start of the experiment.

Presence of betaine in germination medium showed no response on seedling growth of non-salinised or salinised seedlings with 0.1 M salt. However, at 0.15 and 0.2 M salt levels, betaine at 1 or 2 g/L resulted in a very significant increase in shoot length compared to nonbetaine treated and salinised treatments (FIG. Beneficial effect reaches a peak at 2 g/L and further levels actually reduce the shoot growth at both salinities.

1.5 Wheat qernnination in saline soil when betaine is added to the soil The experimental protocol and conditions of plant growth are similar to the one described in the section 1.3 except that in each pot, unimbibed and untreated seed were planted with betaine at 0, 1, 2, 4, and 8 g/L in all combinations with 0, 0.1 and 0.15 M salinity. Seedling emergence was noted every day for 10 days.

The results of the previous petri dish experiments were tested in pot situation. In non-saline soil the percentage of germination was 87.5% and this was drastically reduced to 42% and 35% in response to salinity of 0.1 and 0.15 M respectively. Addition of 1 or 2 g/L of betaine significantly increased germination to almost non-saline conditions at 0.15 WO 95/35022 PC'I/AU95/00357 11 M salinity (FIG. However, any higher levels of betaine seem to have reduced germination at 0.2 M salinity.

1.6 Effect of betaine on water stressed wheat seedlinqs in petri dishes Betaine solution of 0, 1, 2, 4, 8, 16, and 20 g/L was mixed in PEG (MW 4,000) solution. Each petri dish with 10 wheat seed was treated with one of the concentrations of betaine in 12 ml of 20% PEG solution and germination test was performed and shoot and root lengths were measured 10 days after the initiation of the experiment.

Presence of 1 or 2 g/L of betaine in PEG medium significantly increased growth of water (PEG) stressed seedlings. Any higher levels of betaine actually reduced the growth of seedlings significantly (FIG. 6) 1.7 Effect of various betaines on the growth of wheat seedlings Glycinebetaine N-methyl proline N-dimethyl proline or stachydrine(S), N-methyl-trans-4-hydroxy-proline (MHP), Ndimethyl-trans-4-hydroxy-proline (DHP), Trigonelline were the six betaine/betaine analogues tested in this experiment. The compounds were applied at 10, 20 and 40 mM. 12 ml of this solution was placed in petri dishes with 10 wheat seed as described in the section 1,2, Shoot and root lengths were measured after ten days, Only one salinity concentration of 0.2M was used.

Six different betaines/betaine analogues, including glycinebetaine, were tested on the shoot and root growth of 0.2 M salinised wheat seedlings in petri dishes. Only N-methyl proline at 10 mM showed about 11% increase in root length, Trigonelline showed reduced root length by about 13%. N-methyl proline showed no response on shoot length. At this salinity, the other four betaine analogues increased shoot length by as much as 68% compared to control, however, the concentration required by each compound to obtain this effect varied from WO 95/35022 PCT/AU95/00357 12 10-40 mM (Table 3).

2. COTTON 2.1 Effect of betaine on the germination and growth of salinised cotton in pots Plastic pots fitted with plastic lining bags to cover the drainage holes were filled with 1.8 kg Samford loam. Betaine at 0, 1, 2 and 4 g/L was mixed in all combinations with salinity levels of 0, 0.1, 0.15 and 0.2 M. 400 ml of the solution with each of the combinations of betaine and salt was applied to each pot. Ten unimbibed and untreated cotton (Gossypium hirsutum cv. Siokra) seed were planted in each pot.

The experiment was conducted in naturally lit glasshouse (Conpol) at Samford with temperature control (max 35°C min 20°C). Germination counts were taken for 10 consecutive days. Later, pots were thinned to 2 plants /pot where possible and fertilised with 100 ml of Aquasol solution (8 Pots were weighed on alternative days and the lost water was added to 400 ml to maintain the salinity level relatively constant.

Two months after planting, all the leaves in each pot were harvested to measure leaf area using automatic leaf area meter. Stem, and root lengths and the oven dry weights of leaf and shoot (stem leaf wt) were measured.

Non-salinised cotton showed a percentage of germination of above 85%. Salinity had a drastic effect on germination, reducing it to 77%, 37% and 2.5% in response to 0.1, 0.15 and 0.2 M NaCI respectively. At all levels of salinity, betaine increased germination (FIG.

7) compared with non-betaine treated controls. At 0.1 M salinity, betaine application at 1 g/L restored germination back to the non-salinised control level. At 0.15 M and 0.2 M NaCI, 2 g/L of betaine increased germination more than non-betaine treated salinised treatments. At these salinities, any further increase in betaine level showed the tendency of reducing germination from its peak value at the lower betaine levels.

Leaf area of cotton (FIG. 8) was significantly increased by WO 95/35022 PCT/AU95/00357 13 about 20% in non-salinised controls and at 0.1 M salinity, in response to 1 g/L betaine application. At the highest salinity (0.2 M NaCI) the leaf area was increased by more than 3 and 6 fold in response to 1 and 2 g/L betaine application, respectively, Leaf dry wt (FIG 9) also followed similar trends in response to betaine application in saline soil. Shoot dry wt (FIG. 10) was increased in control as well as in 0.15 M salinity by about 40%. At the highest salinity (0.2 M NaCI) dry matter was increased by about 5 times in response to 2 g/L of betaine. Shoot and root lengths were not influenced by betaine application.

2.2 Effect of betaine on water stressed cotton.

Plastic pots fitted with plastic liner bags were filled with kg of Samford loam. 900 ml of solution containing 0, 2, 4, 8 and 12 g/L of betaine was applied to the pots. Ten cotton (cv. Siokra) seed were planted and then the seedlings were thinned down to 5 after germination.

When seedlings were about 4 weeks, 200 ml of Vermiculite was added to the soil surface in each pot. Final watering was done to make sure that each pot contained 900 ml of water and then further watering was stopped. Four weeks after this, relative water content of the youngest and most expanded leaf was measured.

When water was withheld from cotton seedlings, control plants reached to about 40% relative water content (RWC) which is very close to the death point for some crop plants. On the other hand, betaine application from 2 to 12 g/L increased the RWC in a linear fashion to about 75% (FIG. 11) which is similar to the value that plants under moderate stress will contain. This suggests that betaine increases drought tolerance or postpones the occurrence of severe water stress symptoms.

2.3 Effect of betaine on water use and growth of cotton Plant culture for this experiment is exactly same as the one described in the section 2.2. Pots were weighed once in 2 or 3 days to measure water use. In this experiment there were two levels of watering: WO 95/35022 PCTIAU95100357 14 when water content reaches 0.1 kg/pot, well watered plants received watering to field capacity (water content of 0.9 kg/pot); and plants receiving limited watering were maintained only to 0.4 kg of water/pot when the water content fell to 0.1 kg/pot. Care was taken that vermiculite was added to the pot soil surface to cut down evaporation, Blank pots without plants were also maintained to estimate the unavoidable evaporation from the vermiculite surface. Evaporative loss has been deducted from the gross water use of plants to arrive at the net water used in transpiration.

When the plants were 2 months old, shoot length and weight, root length and weight, and leaf area of the plants were measured.

When plants were well watered watered to field capacity when reached wilting point), betaine application at 4 g/L actually resulted in more water consumption than controls for 4 days (FIG. 12) and with the limitation of water availability, water use has started declining than non-betaine treated controls. Betaine application at 8 and 12 g/L resulted in significantly lower water use throughout the period and reaching about 48% less water use compared with the control. Similar pattern of water use was also noted in limited water application treatment also (FIG. 13).

Betaine application reduced shoot length in both well- and limited-watering treatments (Table 4) on a concentration dependent manner. Although shoot weight was also reduced in a similar pattern to the shoot length, the shoot length reduction was to a lesser magnitude.

(Table Root length was not affected by betaine application (data not included), however, root weight of both well- and limited-watered plants was reduced on a concentration dependent manner (Table This suggests that root diameter has reduced in response to betaine application. Leaf area also reduced in both the watering treatments in response to betaine application. (FIG. 14).

3. DESMANTHUS WO 95/35022 PCIT/AU95/00357 3.1 Effect of betaine on Desmanthus virqatus in petri dishes Desmanthus virgatus seed was scarified in a mechanical scarifier fitted with a coarse sand paper. The process was repeated until the seed was no longer glossy and had scars on the seed coat. Scarified seed was treated with glycinebetaine whereby one gram of scarified seed was soaked in 10 ml of one of several glycinebetaine solutions.

Glycinebetaine solutions containing 0, 2, 4, 8 and 12 gram glycinebetaine per litre of distilled water (corresponding to 0, 17 x 10.

2 M, 3.4 x 102 M, 6.8 x 10- 2 M and 1.0 x 10 M respectively) were used. The seeds were soaked for 12 hours at 25°C then immediately dried in a dehydrator at for about 3 hours.

Ten treated seeds were placed in a petri dish containing a base of filter paper. The treated seeds were watered with 13 ml of distilled water or 0.15 M NaCI solution. The treated seeds were incubated in an incubator at 300C for 4 days in the dark before the length of the root (radicle) and shoot (plumule) were measured. The germination count of the seeds was above 90% when distilled water was used as the germinating medium in the petri dishes. The growth of seedlings as indicated by the length of the shoot and root is shown in FIG Radicle length increased by more than three-fold with seeds treated with 12 g/L glycinebetaine compared with non-treated seeds when watered with either distilled water or 0.15 M NaCI solution. The beneficial effects of betaine was shown to be not dependant on salinity alone.

The shoot length of 12 g/L glycinebetaine treated seeds was approximately 1.6 times the shoot length of non-treated seeds when watered with either distilled water or 0.15M NaCI, However, the shoot length of 12 g/L glycinebetaine treated seeds watered with distilled water was approximately 1.3 times the shoot length of similarly treated seeds but watered with 0.15 M NaCI. It should be noted though that the shoot length of 12 g/L glycinebetaine treated seeds watered with 0.15 M saline was longer than the shoot length of non-treated seeds watered with WO 95/35022 JIMAIJ95/0357 16 distilled water.

One conclusion from the experiment was that seeds treated with 12 g/L glycinebetaine by soaking for 12 hrs at 25 0 C and watered with 0.15 M NaCI can germinate in what would be normally growth restrictive conditions.

3.2 Effect of betaine with soaking of Desmantus virgatus seeds for various times Desmanthus virgatus seed was scarified in a mechanical scarifier fitted with a coarse sand paper. The process was repeated until the seed was no longer glossy and had scars on the seed coat. Scarified seed was treated with glycinebetaine whereby one gram of scarified seed was soaked in 10 ml of one of several glycinebetaine solutions.

Glycinebetaine solutions containing 0, 12, 24 and 36 gram glycinebetaine per litre of distilled water (corresponding to 0, 1.0 x 10' 1 M, 2.0 x ld M and 3.0 x 10' 1 M respectively) were used. The seeds were soaked for 2 or 4 hrs at 25°C then immediately dried in a dehydrator at 45°C for about 3 hrs.

Ten treated seeds were placed in a petri dish containing a base of filter paper. The treated seeds were watered with 13 ml of distilled water or 0.15 M NaCI solution. The treated seeds were incubated at 30 0 C for 4 days in the dark before the lengths of the root (radicle) and shoot (plumule) were measured. The germination counts of the seed was above 90% when distilled water were used as the germinating medium in the petri dishes. The results of the experiment are shown in Tables 6 and 7.

Seeds that were soaked in the various betaine solutions for 2 hrs did not show significant differences in root length, shoot length and seedling weight during germination compared with controls, This was observed when the seeds were watered with distilled water or 0.15 M NaCI.

Seeds that were soaked for 4 hrs in betaine solutions of 12 g/L and 24 g/L betaine produced shoots that were longer and seedling WO 95/35022 PCT/AU95/00357 17 weights that were greater than the corresponding controls when the seeds were watered with 0.15 M NaCI. The germination of seeds soaked for 4 hours in 36 g/L betaine solution appeared to be inhibited.

3.3 Effect of betaine on germination of salinised Desmanthus virqauts in pots Six inch plastic pots fitted with plastic bags and filled with 1.8 kg Samford loam were applied with 450 ml of solutions containing betaine at 0, 1, 2 and 4 g/L in all combinations with 0, 0.05, 0.1 and 0.2 M salt. 20 unimbibed, untreated and scarified seed were planted and water content maintained at 450 ml/pot by weighing them on alternative days for two weeks. Germination and emergence were monitored during this period.

The effect of betaine was significant only at the salinity concentration of 0.1 M (FIG. 16). Germination increased from 17% in non-betaine treated salinised treatment to 31% in response to betaine application at 1 g/L. Higher betaine application rates actually reduced germination from its peak at 1 g/L betaine application.

4. TOMATO 4.1 Effect of betaine on the growth of tomato seedlings Six inch plastic pots fitted with plastic bags and filled with 1.8 kg Samford loam were applied with 350 ml of solutions containing betaine at 0, 1, 2 and 4 g/L in all combinations with 0, 0.025, 0.05 and 0.075 M salt, 20 unimbibed and untreated tomato (Lycopersicon esculantum cv. Grosse Lisse) seed were planted and water content maintained at 350 ml/pot by weighing them on alternative days for two weeks. Germination and emergence were monitored during this period.

Then the seedlings were thinned to 5 plants per pot and shoot dry wt was obtained 2 months after planting.

Betaine showed no significant increase in germination of tomato seed subject to 0, 0.025, 0.05 and 0.075 M salinity. However, the seedling dry weight was increased at all levels of salinity at betaine PCr/AU 9 5 0 H0 i Z RECEIVED 22 MAY 18 application rates of 1 and 2 g/L and further higher application of betaine showed the reducing tendency of shoot dry weight (FIG. 17).

Field trials using seed treated with glycinebetaine: 5.1 Seed treatment: with betaine: Methyl cellulose solution was used as a sticker to hold glycinebetaine on the seed surface. Five g of methyl cellulose powder was dissolved in 100 ml of boiling water to prepare 0.5% strength methyl cellulose. When methyl cellulose was completely dissolved, the solution was cooled and left in the fridge over-night. Fifty grams of this methyl cellulose solution was sprayed on to cotton seed while seed was being rotated in a long beaker.

Mixing continued until a uniform coating was obtained and the layer dries to become sticky and just moist.

Complete drying was avoided as it may lead to loss of stickiness of methyl cellulose layer. Finely ground glycinebetaine was sprinkled 0.0, 2.5, 5.0, 7.5 and 10.0% on methyl cellulose coated seed. It is advisable to add required amount of glycinebetaine in several steps, instead of in one dose, to obtain uniform glycinebetaine coating. Glycinebetaine is highly hygroscopic and to dry the seed surface, 50 g of dried and powdered peat/kg of the coated seed was added in several increments. Any other inert and dry powder could be used to replace peat.

5.2 Field germination of cotton: from seed treated with betaine: Salinity affected cotton growing field [0-10 cm depth EC(saturation) was 14 mScm- 1 near Dalby in Queensland state of Australia was selected to field test effect of glycinebetaine seed treatment on germination and seedling vigour. Seed was treated as described in section 2.1.1 with glycinebetaine 0, 2.5, 5.0 and w/w and sown in soil with moisture content of S stafflhll oy/koep/PCT.AU95_1 22.5.96 a. o: AMENDED SHEET

IPEA/AU

cr/Au 95 0 0 3 57 RECEIVEU 1 9 JUL 1996 18/1 Fifty seeds from each treatment lot were sown in a 2m long row of 2 x Im plot to a depth of 5 cm. All treatments were replicated 3 times in completely randomised block design. Seedling emergence was counted 3 weeks after planting. At the same time, all seedlings were cut at the collar region and dried in an airforced oven at 60 0 C for 24 h before dry weights were recorded.

5.3 Field experiments with betaine treated seed and foliar application: This experiment was conducted on a non-saline soil near Dalby to test the effect of seed treatment and to find if the crop responds to any booster doses of glycinebetaine by foliar applications. Cotton seed of cv. V2 was treated with glycinebetaine 0, 2.5, and 7.5% in the same way as described in 2.2.1.

One set of treatments received only seed treatment.

The second set of seed treated plots received o-glycinebetaine foliar application 2kg/ha at 2 weeks after emergence. The third set of seed treated plots received two foliar applications at 2 weeks after emergence and at square formation stage.

Conclusion: Figure 18 depicts the increase in cotton germination in saline field conditions due to betaine treatment of the cotton seeds.

Germination percentage of about only 50% was obtained (Fig 18) when cotton seed with a germination capability of about was sown in saline field conditions with an EC (saturation) of 14 mScm' 1 Glycinebetaine treated seed or 5.0% were able to maintain the germination to well above 80%. However, any further dose of betaine was not beneficial and germination at high level of betaine was similar tp that in control.

Figure 19 depicts the increase in cotton seeding growth in saline field conditions due to betaine treatment of the 10.7 AMENDED SHEET

IPEA/AU

c U 9 5 0 0 3 5 7 RECEIVED 1 9 JUL 1996 18/2 cotton seeds.

The dry matter of seedlings treated with betaine 5% was the highest and it was about 40% greater than the seedlings resulted from untreated plants (Fig. 19). Similar to germination, 7.5% betaine seed treatment showed no increase in dry matter compared to control.

Figure 20 depicts seed-cotton yield (kg/ha) as influenced by betaine seed treatment and foliar applications.

There was a significant increase in seed-cotton yield in response to 5 and 7.5% betaine seed treatment, which was equal to 18 and 22% increaseover control, respectively (fig. 20). Foliar applications did not increase yield when seed were not treated with glycinebetaine. However, its combination with 2.5 of 5.0% betaine seed treatment was beneficial. Foliar application for seedlings that were treated with 7.5% betaine showed an adverse response. This adverse response was more dramatic when two foliar applications were applied.

10.7 i; FLL AMENDED SHEET

IPEA/AU

wo 95/35022 P107A U9510039 RECEIVED 2 2 MAYk 19 TABLE I NAMES OTHER NAMES Glycinebetaine Oxyneurin 1-aaninebetaine Homobetalne 2-trimethylamino-6-ketoheptanoate Prof inebetaine Stachydrine Proline N-methyl-L-proline Trans-4-hydroxy-N-methyl-L-proline C/s-3-hydroxy-N-methyl-L-proflne (-)4-hydroxyproIin ebeta ine Betonicine (+)4-hydroxyprolinebetaine rrcn 3-hydroxyprolinebetaine 3-oxystachydrine Histidinebetaine Herzynine, Ercinine Try ptophanbetaine Hypaphorine 2-mercaptohistidine-betaine Ergothioneine Pipecolatebetaine Homostachydrine Nicotinic acid betaine Trigonelline 2j AMENDED SHEET

JPEAIAU

WO 95/35022 WO 9535022PCT/A195/00357 TABLE 2 Time of Root length Shoot length Water uptake Soaking (cm) (CM) (g/g seed) 0 6.9 3.2 0 2 6.6 3.2 2 4 6.3 3.2 6 8 1 6.6 3.2 9 12 6.4 3.1 9 TABLE 3 Treatment 10 mM 20 mM 40 mM Root Shoot Root Shoot Root Shoot Control 1.5 1.8 1.5 1.8 1.5 1,8 Glycinebetaine 1,5 2.5 1.5 2.3 1.5 N-methyl 1.7 1.8 1.5 1.7 1.6 1.8 prolime N-dimethyl 1.5 1.6 1.4 1.6 1.5 3.1 proline or stachydrine N-methyl-trans- 1.4 2.7 1.5 2.7 1.4 2.9 4-hydroxyprol ine__ N-dimethyl- 1.5 2.3 1.5 3.0 1.5 2.2 trans-4hydroxy-proline IT 3_ 1__1.9 TrIgonelline 1.3 16 .3 28 1. 1.

WO 95/35022 PCTAU9$/00357 TABLE 4 Shoot length (cm) Shoot wt (g/5 plants) Betaine Well- Limited Well- Limited watered watering watered watering 0 21.2 16.5 1.88 2.14 4 20.2 15.7 1.64 1.93 8 18.8 14.7 1.47 1.72 12 16.7 14.02 1.58 1.59 TABLE Betaine Well-watered Limited watering 0 1.1 0.9 4 1.0 0,9 8 0.8 0.8 12 0.5 0.8 WO 95/35022 PCT/U95/00357 TABLE 6 TWO HOURS SOAKING Parameter NaCI Glycinebetaine level (g/L) concentration 0 12 24 36 Root length 0.0 M 2.6 2.8 2.6 (cm) )0.15 M 2.1 1.7 1.5 1,3 Shoot length 0.0 M 3.8 3.9 3.8 3.3 (cm) 0.15 M 1.5 1.5 1.4 1.3 Seedling 0.0 M 0.23 0.27 0.23 0.24 weight (g/10) w t 0.15 M 0.20 0.20 0.21 0.21 TABLE 7 FOUR HOURS SOAKING Parameter NaCI Glycinebetaine level (g/L) concentration 0 12 24 36 Root length 0.0 M 2.7 2.4 2.6 (cm) 0.15 M 1.8 1.5 1.6 1.4 Shoot length 0.0 M 3.8 3.5 3.9 3.7 (cm) (c 0.15M 0.9 1.6 1.7 1.2 Seedling 0.0 M 0.26 0.24 0.29 0.30 weight (g/10) 0 0 0 0,15 M 0.16 0.23 0.19 0.11

I

WO 95/35022 PCr/A1J95100357 23

LEGENDS

TABLE 1 List of major betaines.

TABLE 2 Effect of soaking time on wheat seedling root and shoot lengths and water uptake imbibition.

TABLE 3 Effect of different concentrations of betaine/betaine analogues on root and shoot growth of wheat seedlings at 0.2 M salinity.

TABLE 4 Effect of betaine and watering regimes on shoot length (cm) and shoot weight (g/5 plants).

TABLE Effect of betaine and watering regimes on root weight (g/5 plants).

TABLE 6 Germination after 4 days at 30 0 C of Desmanthus virgatus seeds previously soaked in a glycinebetaine solution for two hours TABLE 7 Germination after 4 days at 30"C of Desmanthus virgatus seeds previously soaked in a glycinebetaine solution for four hours FIG. 1 Betaine imbibition on shoot growth of wheat in petri dishes FIG. 2 Effect of betaine imbibition on wheat germination in saline soil FIG. 3 Grain yield of salinised wheat in response to seed soaking in betaine FIG. 4 Effect of betaine on shoot growth in saline medium of wheat in petri dishes FIG. Effect of betaine on germination of wheat in saline soil WO 95/35022 lPCT/AU95/00357 24 FIG. 6 Effect of betaine of PEG on shoot growth of wheat FIG. 7 Betaine in saline soil and cotton germination FIG. 8 Betaine in saline soil and cotton leaf area FIG. 9 Betaine in saline soil and cotton leaf dry wt FIG. Betaine in saline soil and cotton shoot dry wt FIG. 11 Drought survival or relative water content (RWC) of cotton FIG. 12 Betaine reduces water use of well-watered cotton seedlings FIG. 13 Betaine reduces the water use of cotton plants with limited watering FIG. 14 Reduction of leaf area of well- and limited- watered cotton plants in response to betaine application FIG. Effect of betaine seed soaking and growth of Desmanthus virgatus in 0.15 M NaCI solution FIG. 16 Betaine and salinity influencing germination of Desmanthus in soil FIG. 17 Dry matter accumulation of tomato seedlings in response to salinity and betaine application

Claims (9)

1. A method for treating a seed to enhance seedling growth and/or protect against environmental stress during germination by treating the seed with betaine prior to planting whereby the seed is immersed in a solution of the betaine and said solution has a 0.34 M or lower betaine concentration, or the seed is coated with a solid form of the betaine at a ratio of 1-10 betaine weight per seed weight.

2. A method as claimed in Claim 1 wherein the seed is a cereal seed, cotton seed or a seed of any other commercially significant crop.

3. A method as claimed in Claim 1 wherein the method includes one or more coatings of an adhesive and/or a drying agent and/or the betaine is glycinebetaine.

4. A seed treated with betaine whereby the seed is immersed in a solution of the betaine and said solution has a 0.34 M or lower betaine concentration, or the seed is coated with a solid form of the betaine at a ratio of 1-10 betaine weight per seed weight.

A seed treated by betaine whereby the seed is coated with the betaine at a ratio of 1-10 betaine weight per seed weight and one or more coatings of an adhesive and/or a drying agent.

6. A seed as claimed in Claim 6 wherein the adhesive is methyl cellulose or gum arabica and the drying agent is lime.

7. A seed treated by betaine whereby the seed is immersed in a solution containg 0.34 M or lower betaine concentration and one or more coatings of an adhesive and/or a drying agent.

8. A seed as claimed in Claims 4, 5, 6 or 7 wherein the betaine is glycinebetaine.

9. A seed as claimed in Claims 4, 5, 6, 7 or 8 wherein the seed is a seed from a commercially significant crop. A wheat seed treated with betaine wherein the seed is immersed in a solution containing 0.1-0.34 M glycinebetaine.