FIELD OF THE INVENTION

The present invention relates to agricultural technology relating to methods for developing waxy starch cassava varieties with improved properties and lower cyanide content.

BACKGROUND OF THE INVENTION

In addition to the use of normal starch, which consists of amylose and amylopectin, nowadays amylopectin starch (which has no amylose) is being extensively employed as a thickener, stabilizer, texturizer, viscosity modifying agent, adhesive, binder and coating agent in several industrial applications that require high starch paste stability. For instance, in the production of some food products, paper, textile and personnel care products, the use of amylopectin starch is preferred due to its characteristic of showing no or -low retrogradation. However, starch containing only amylopectin, also known as waxy starch, can be found only in some cereals such as rice, corn, sorghum, barley and wheat, or tubers such as.potato and cassava, which have the homozygous recessive genes, i.e. wxwx. This homozygous recessive genes which lead to the synthesis of waxy starch occur through the mutation of the gene that regulates the amylose-synthesizing enzyme, i.e. granular bound starch synthase (GBSS).

The production of waxy corn starch has greatly increased in recent years, and its price is relatively much higher than that of regular corn starch. Nevertheless, the production of waxy corn starch can be contaminated by the grain of regular corn starch varieties. It is, therefore, important to grow waxy corn separate from regular corn to prevent cross-pollination and contamination, which means that more planting area is required. In contrast, .this problem hardly occurs when waxy starch cassava is grown as cassava is propagated asexually and does not depend on the sexual seed (or grain) for yield. Contamination can be circumvented by preventing the mixing of waxy starch cassava roots with normal cassava roots at harvest.

Cassava is one of the most important economic crops of the world. In Thailand, cassava is widely grown, and products derived from cassava have been and are being exported for various applications, in particular in food products. Examples of important cassava products are chips, pellets, native starches, modified starches, pearls and sweeteners. Important attributes of normal cassava starch and waxy corn starch are compared; while some of these are comparable, the extent of starch retrogradation is quite different, as summarized in Table 1.

Table 1. Comparison of important characteristics of waxy corn starch and normal cassava starch

Accordingly, there is interest to develop cassava varieties with waxy starch, which can lead to the development of specialty starch products and open up new markets for specific applications. This can help add more economic value to cassava starch and further enhance its export market.

One disadvantage of most cassava varieties grown in Thailand is the accumulation of cyanide, in a form of cyanogenic glucoside, in the storage roots. During cassava starch production, the roots must be processed properly to remove this toxic compound so that the processed starch contains very low cyanide content, complying with the safety regulations, particularly the food safety regulation. When consumed, the residuals of the cyanogenic compound in starch can be transformed into hydrocyanic acid by enzymes in the small intestines and become toxic (LD ₅ 0 = 1-3 mg per kg body weight). The objectives of cassava variety development are not only to improve yield characteristics, but also to reduce the content of cyanogenic compound in the roots for safe use and to reduce the processing cost for cyanide removal.

There are many prior art documents demonstrating the effect of inhibiting GBSS on starch composition. For instance, U.S. Patent No. 6,784,338 discloses the process of preventing amylose synthesis by eliminating the GBSS activity in potato, with the transformation of antisense constructs to the gene encoding said GBSS enzyme. Amylopectin starch in potato is thus obtained. U.S. Patent Application Publication No. 2007/0261136 discloses the method of making amylopectin corn starch by transformation of a chimeric nucleic acid sequence encoding waxy starch. The disclosed methods in these aforementioned prior art s apply genetic modification (GM) or transformation techniques, which still have limited consumer acceptability in view of concerns over safety as well as ecological and environmental issues.

One potential method for the development of non-GM cassava variety with a recessive mutation for the waxy trait is by the inbreeding method, using self-pollination. This has been reported when an amylose-free starch cassava mutant, AM 206-5, was discovered by Ceballos et al. (2007) (Ceballose et al., 2007, Discovery of an amylose-free starch mutant in cassava (Manihot esculenta Crantz), J. Agri. Food Chem. 56: 7215-7222). This clone, when planted, however, gave very low root yield, and because of its small plant stature is not suitable for commercial purpose. SUMMARY OF THE INVENTION

The present invention aims to develop cassava varieties with desirable traits, in this case waxy starch cassava with improved properties and low cyanide content, by using non-GM techniques.

Thus, the present invention involves a method of developing waxy starch cassava varieties with improved properties and low cyanide content. The method comprises crossing a low-yielding waxy starch cassava genotype (wxwx) with normal cassava varieties (which are homozygous dominant, WXWX). The selected normal cassava varieties should have good phenotypic traits including agronomic traits, plant growth form, root yield and adaptability to the similar environment with that of Thailand, with one or more additional traits such as high tolerance to environmental stress, high root productivity, high harvest index and high dry matter content of fresh roots. The crosses result in F[ hybrids with the gene combination, WXwx. F ₂ progenies are then obtained by cross-pollinations between F] hybrids (F _\ x Fi); these will bear the homozygous recessive genes, wxwx, as well as express desirable traits, namely, waxy starch cassava with improved properties and low cyanide content. Furthermore, the present invention relates to the cassava varieties resulting from the breeding technique stated above. In addition, the present invention encompasses the starch obtained from the waxy starch cassava varieties with improved properties and low cyanide content, developed by the method described above.

The present invention also relates to the applications of the starch obtained from these waxy starch cassava varieties, developed by the method described above, in various products and industries such as food and beverages, feeds, papers and packages, adhesives and binders, coating agents, textile goods and clothes, pharmaceutical products, cosmetics, personal care products, hair care products, pet care products and household products.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 , Paste viscosity profile of cassava starches extracted from different cassava varieties, including T WX 1 , TKWX 2, TKWX 3, AM 206-5 and Huay Bong 60

Figure 2. Degree of syneresis (%) of cooked pastes of waxy corn starch, waxy rice starch, waxy potato starch, waxy cassava starch obtained in the present invention and normal cassava starch at different freeze-thaw cycles

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, the term "waxy starches" refers to starches that contain very low amylose content and very high amylopectin content, including starches with amylopectin content more than 90%, preferably those with amylopectin content more than 99%, and more preferably those with 100% amylopectin content or 0% amylose.

In the present invention, the term "cassava varieties with improved properties" refers to cassava varieties having one or more of the desired properties such as higher root productivity, higher number of roots per plant, containing waxy starches with specific attributes, e.g. higher paste viscosity, paste clarity, freeze-thaw stability as well as lower amylose contamination, when compared to their parents.

In the present invention, the term "lower cyanide content" means that roots or starches obtained from the varieties developed under the present invention contain lower cyanide content than normal cassava varieties.

In the present invention, the term "harvest index" is the numeric value obtained as the ratio between the storage root weight and the total plant weight, i.e. the weight of the plant parts above the ground plus the weight of the roots. The breeding of cassava varieties under the present invention begins by cross-pollinations between the low-yielding waxy starch cassava mutant and normal cassava varieties to transfer the waxy trait to the Fi hybrids. The F hybrids are then crossbred with other F _t hybrids to get F ₂ progenies. The cross-pollinations between the waxy starch cassava mutant and normal cassava to generate Fi hybrids can segregate the waxy recessive trait; therefore, normal cassava varieties with any remarkable traits that can be selected from the group consisting of high tolerance to environmental stress, high root productivity, high harvest index and high dry matter content of the fresh roots can be used for producing the Fj hybrids..

As the required trait/s in the F ₂ progenies can be fixed by subsequent cloning, breeding of desired genotypes can be accomplished, and is not limited to the generated Fi hybrids themselves. Alternatively, this can be done by crossing Fi hybrids with another Fi hybrids generated from crossing waxy starch cassava and normal cassava, having similar or different traits; or by crossing heterozygous F _! hybrids with hybrids obtained from open breeding of the waxy mutant; or by crossing heterozygous Fi with open-pollinated plants of waxy female parents.

F ₂ progenies with waxy starch can be differentiated from normal starch by (but not limited to) iodine staining or DNA fingerprinting. Other known methods to prove that the generated genotypes have waxy starch can be also applied to check for the F ₂ progenies with waxy starch. In addition, this can be proven by growing F ₂ progenies in the field and then collecting the roots for starch isolation.^ The properties of isolated starch are analyzed, and F ₂ hybrids that give good starch properties and low cyanide content are then selected for further planting and evaluation in suitable cassava-growing areas.

The starches isolated from waxy starch cassava varieties obtained by the method described in the present invention have one or more of these improved properties, selected from the group consisting of low or no retrogradation, high stability of paste clarity, high paste viscosity and low shear resistance of the cooked paste.

Accordingly, isolated starches can be applied in many industries, such as, but not limited to food and beverage, feed, paper and packaging, adhesive and binder, coating, personal care products, hair care products, pet care products and household products.

EXAMPLES

The following examples described herein are provided in order to clarify the detailed description only and are not intended to the scope of the present invention. Example 1. Development of waxy starch cassava variety TKWX 1

Two groups of Fi hybrids were generated and used as the parent lines. The first group of Fi hybrids, used as the female parent, was obtained by crossing waxy starch cassava clone, AM 206-5, with a normal cassava clone, MTAI 8 (having high tolerance to environmental conditions and high root productivity), through cross pollination. The other group of Fi hybrids, used as the male parent, was developed by cross-pollinating through open-pollination (i.e. with unknown pollen source) of the female flowers of AM 206-5. ^ These two Fi hybrids then were intercrossed to generate the F ₂ progenies.

Seeds of the F ₂ progenies were produced and then used for planting in the field of the Thai Tapioca Development Institute, located at Huay Bong District, Nakhon Ratchasima, to select for the waxy starch cassava genotypes with the best agronomic properties. This resulted in TKWX 1 (ΗΒ^09-989-9), as shown in Table 2.

Table 2. Agronomic traits of waxy starch cassava, TKWX 1 (HB _wx 09-989-9).

Trait TKWX 1 (HB _wx 09-989-9)

1. Growth form Erect

2. Number of stems/plant 4

3. Leaf scars on nodes Semi- prominent

4. Growth habit Erect

5. Apical leaf color Purplish green with pubescence

6. Shape of central leaflet Lanceolate

7. Color of leaf veins Green

8. Length of petiole Moderate (21.3 cm)

9. Orientation of petiole < 60° (41.4°)

10. Petiole color Reddish green

1 1. Number of leaf lobes 9

12. Length of leaf lobes 16-20 cm

13. Width of leaf lobes 3.6-4.5 cm

14. Stem color Orange Trait TKWX 1 (HB _wx 09-989-9)

15. Plant height 201 -250 cm

16. Direction of root formation Vertical

17. Root shape and skin condition Conical with rough skin

18. Color of root exterior Dark brown

19. Color of root cortex Cream

20. Color of root flesh White

21. Exterior peel removal Easy

22. Ease of external peel removal Easy

23. Number of roots/plant 6-10

Example 2. Development of waxy starch cassava variety TKWX 2

Two groups of Fi hybrids were generated and used as the parent lines. The group of first Fi hybrids, used as the female parent, was obtained by crossing the waxy starch cassava clone, AM 206-5, with a normal cassava clone, SM 1219-9 (having high tolerance to environmental conditions, high root productivity and high harvest index), through cross pollination. The other group of Fi hybrids, used as the male parent, was developed by cross-pollinating of the waxy starch clone, AM 206-5, with another normal cassava clone, CM 3306-4 (having high root dry matter content and low cyanide content). These two Fi hybrids were then crossed to generate the F ₂ progenies.

Seeds of the F ₂ progenies were produced and then used for planting in the field of the Thai Tapioca Development Institute, located at Huay Bong District, Nakhon Ratchasima, to select for the waxy starch genotypes with the best agronomic properties, resulting in TKWX 2 (ΗΒ„χ09- 19-2), as shown in Table 3. Table 3 Agronomic traits of waxy cassava, TKWX 2 (116^09-19-2).

Trait TKWX 2 (HBwx09-19-2)

1. Growth form Spreading

2. Number of stems/plant 4

3. Leaf scars on nodes Semi- prominent

4. Growth habit Straight

5. Apical leaf color Light green with pubescence

6. Shape of central leaflet Pandurate

7. Color of leaf veins Green

8. Length of petiole Moderate (22.8 cm)

9. Orientation of petiole Inclined upwards < 60° (45.8°)

10. Petiole color Reddish green

11. Number of leaf lobes 9

12. Length of leaf lobes 20-25 cm

13. Width of leaf lobes 2.0-2.4 cm

14. Stem color Brownish green

15. Plant height 201-250 cm

16. Direction of root formation Vertical

17. Root shape and skin condition Conical with rough skin

18. Color of root exterior Dark brown

19. Color of root cortex Cream

20. Color of root flesh White

21. Exterior peel removal Easy

22. Ease of external peel removal Easy

23. Number of roots/plant 10-15 Example 3. Development of waxy starch cassava variety TKWX 3

Two groups of Fi hybrids were generated and used as the parent lines. The first group to be used as the female parent, was obtained by crossing the waxy starch clone, AM 206-5, with a normal cassava clone, MCOL 1505 (having high root dry matter content), through cross-pollination. The other group, to be used as the male parent, was developed by cross-pollinating the waxy starch AM 206-5 with another normal cassava, CM 3306-4 (having high root dry matter content and low cyanide content). These two Fi hybrids were then intercrossed to generate the F ₂ progenies.

Seeds of the F ₂ progenies were produced and then used for planting in the field of the Thai Tapioca Development Institute, located at Huay Bong District, Nakhon Ratchasima, to select for the waxy starch genotypes with the best agronomic properties, resulting in TKWX 3 (ΗΒ^09- 635-4), as shown in Table 4.

Table 4 Agronomic traits of waxy cassava, TKWX 3 (HB _wx 09-635-4).

Traits TKWX 3 (HB _WX 09-635-4)

1. Growth form Erect

2. Number of stems/plant 3

3. Leaf scars on nodes Semi-prominent

4. Growth habit Erect

5. Apical leaf color Purple green with pubescence

6. Shape of central leaflet Lanceolate

7. Color of leaf veins Green

8. Length of petiole Moderate (23.1 cm)

9. Orientation of petiole Inclined upwards < 60° (27,7°)

10. Petiole color Reddish green

11. Number of leaf lobes 9

12. Length of leaf lobes 15-20 cm

13. Width of leaf lobes 4.5-5.0 cm

14. Stem color Orange-green

15. Plant height 201-250 cm Traits TKWX 3 (HB _WX 09-635-4)

16. Direction of root formation Vertical

17. Root shape and skin condition Conical with smooth skin

18. Color of root exterior Dark brown

19. Color of root cortex Cream

20. Color of root flesh White

21. Exterior peel removal Easy

22. Ease of external peel removal Easy

23. Number of roots/plant 6-10

ANALYSIS

Example 1. Comparison of agronomic traits of various cassava varieties

Various cassava varieties including waxy starch varieties, TKWX 1 , TKWX 2, TKWX 3 and AM 206-5 and normal cassava, namely, Huay Bong 80 (having high root productivity), were grown in the same field. At 9 months after planting, plant height, harvest index, length and diameter of storage roots and the number of roots per 5kg were recorded as reported in Table 5. The data suggest that all waxy starch varieties developed in the present invention, i.e. TKWX 1, TKWX 2 and TKWX 3, had improved agronomic properties relative to the female parent, i.e. AM 206-5, and which are comparable to Huay Bong 80, one of the cassava varieties widely grown in Thailand.

Table 5 Some agronomic traits of various waxy starch cassava varieties (TKWX 1, TKWX 2,

TKWX 3 and AM 206-5) and normal cassava, Huay Bong 80

Variety TKWX 1 TKWX 2 TKWX 3 AM-206-5 Huay Bong 80

Plant height (cm) 219 205 210 80 220

Harvest index 0.45 0.49 0.52 0.21 0.60

Length of root (cm) 24 20 22 18 25

Diameter of root (cm) 13 12 13 10 14

No. of roots per 5 kg 18 14 14 24 17 Example 2. Contents of starch, protein and fiber in fresh roots and extracted starch

Determination of starch content in fresh roots

Fresh cassava roots of the different varieties were peeled, cut and ground into small pieces. Around 5 g of each ground sample were hydrolyzed by 20 ml of 25% hydrochloric acid, and the mixture was then filtered through the filter paper. The filtrate was then made up to 500 ml with distilled water. Around 150 ml of this sample was adjusted to pH 7.0 and made up to 250 ml. This solution was then titrated with Fehling's reagent using methylene blue as an indicator. The starch content in the roots was calculated as shown below:

% Starch content ^:

^ Weight of sample (g) x 150 x Volume of solution (ml)

Determination of protein content in extracted starch

Protein content in the starch extracted from the different cassava varieties, namely, TKWX 1, TKWX 2, TKWX 3, AM 206-5 and Huay Bong 80 was determined by the Kjeldahl method. Ground samples were digested with concentrated sulfuric acid and heated in the presence of a catalyst (CuS0 ₄ and K ₂ S0 ₄ 3:97). After complete digestion, the samples were distilled by a nitrogen distillation unit, and then titrated with hydrochloric acid solution. The amount of protein was calculated by the following equation:

% Protein content HC1 concentration (N) x Volume of HC1 (ml) x 1.401 x 6.25 x 100

Weight of sample (g)

Determination of fiber content in extracted starch

Fiber content in the starch extracted from the different cassava varieties (TKWX 1, TKWX 2, TKWX 3, AM 206-5 and Huay Bong 80) was determined by hydrolysis of 100-g ground samples with a-amylase enzyme and heat. The hydrolyzed samples were filtered through filter cloth, washed with hot water and dried. The amount of fiber was calculated as follows: % Fiber content ^: Weight of sample after drying (g) - Weight of filter paper (g) x 100

Weight of initial sample (g) Table 6 Starch content in roots and protein and fiber contents in starches extracted from various waxy starch cassava varieties (TKWX 1, TKWX 2, TKWX 3 and AM 206-5) and normal cassava, Huay Bong 80

Example 3. Iodine testing and determination of amylose content in extracted starch

Starches extracted from the different cassava varieties, TKWX 1, TKWX 2, TKWX 3, AM 206-5 and Huay Bong 80, were tested by staining with 1% iodine solution, and the color of samples was recorded. In addition, the amylose content of the extracted starches was estimated by a spectrophotometric method, according to McGrance et al. (A Simple and Rapid Colorimetric Method for the Determination of Amylose in Starch Products, McGrance, et al., Starch, 1998, 50, 158-163).

The results of iodine testing and amylose determination clearly demonstrate that TKWX 1 , TKWX 2 and TKWX 3 were all waxy starch varieties, as same as their female parent AM 206-5.

Table 7 Iodine testing and amylose content of starches extracted from various waxy starch cassava varieties (TKWX 1, TKWX 2, TKWX 3 and AM 206-5) and normal cassava, Huay Bong 80

Color when tested with

Variety Amylose content (%)

1 % iodine solution

TKWX 1 Reddish brown 0

TKWX 2 Reddish brown 0

TKWX 3 Reddish brown 0

AM-206-5 Reddish brown 0

Huay Bong 80 Dark blue 25 Example 4. Detennination of starch paste viscosity

The paste viscosity of starch extracted from the various cassava varieties, TKWX 1, TKWX 2, TKWX 3, AM 206-5 and Huay Bong 80, was determined according to the method described by Newport Scientific (1995). Starch slurry (6% dry weight basis) was analyzed under the following conditions:

Stirring rate at 960 rpm for the first 10 sec, and then at 160 rpm throughout the test run\ Initial temperature at 50°C for 2min; heating to 95°C at a heating rate of 4.62°C/min and holding for 15 min; cooling to 50°C at a cooling rate of 4.62°C/min and holding for 10 min

Viscosity parameters of the cooked paste were recorded, and these included pasting temperature (°C), peak viscosity (BU), trough viscosity (BU) and final viscosity (BU). The results are presented in Table 8 and Figure 1. All the waxy starches had higher peak and trough viscosities than normal cassava starch.

Table 8 Paste viscosity of various starches extracted from various waxy starch cassava varieties (TKWX 1, TKWX 2, TKWX 3, and AM 206-5) and normal cassava, Huay Bong 80.

Example 5. Determination of starch retrogradation

Starch retrogradation of various starches, including the waxy cassava starches extracted from TKWX 1 , TKWX 2, TKWX 3 and AM 206-5, waxy corn starch, waxy rice starch, waxy potato starch and normal cassava starch from Huay Bong 80, was determined by measuring paste clarity of the cooked starch samples kept for different time durations. Initially, the starch slurry (2% wb, containing 0.02% sodium azide) was gelatinized in a boiling water bath at 100°C, with intermittent stirring at 300 rpm for the first 5 min and then for 1 min every 10 min, until 30 min had lapsed. The cooked paste was then transferred to a cuvette and covered with paraffin film. The paste clarity of freshly cooked paste was measured as % light transmittance (%T) at 650 nm, using 0.02% sodium azide as reference. The sample was then kept at 4°C for 7 days and % light transmittance was recorded after that time. Differences in %T of freshly cooked and refrigerated samples were calculated and summarized in Table 9. Freshly cooked paste of waxy cassava starches produced by the present invention had similar clarity to waxy potato starch, but had higher clarity than waxy corn, waxy rice and normal cassava starches. When kept in a refrigerator, the paste clarity of waxy starches produced in the present invention (from TKWX 1 , TKWX 2 and TKWX 3) was still similar to that of the freshly cooked samples, whereas the paste clarity of the other starches decreased significantly. These results suggest that the cooked pastes of starches extracted from waxy starch cassava varieties had better stability than the other starches, and their stability was comparable to that of their female parent, i.e. AM 206-5.

Table 9 Degree of starch retrogradation, measured as changes in % light transmittance (%T) of fresh (at day 0, Ti) and refrigerated (at day 7, T ₂ ) cooked pastes prepared from various starches, including waxy cassava (TKWX 1, TKWX 2, TKWX 3 and AM 206-5), waxy corn, waxy rice, waxy potato and normal cassava (Huay Bong 80)

Degree of starch

Variety Light transmittance (%T)

retrogradation

At day 0 (%Ti) At day 7 (%T ₂ )

Waxy corn 82.7 65.6 Moderate

Waxy potato 86.5 77.4 Low

Waxy rice 66.4 43.5 Moderate

TKWX1 84.9 84.9 Low

TKWX2 85.7 84.5 Low

TKWX3 83.2 82.6 Low

AM-206-5 85.7 85.3 Low

Huay Bong 80 35.4 0 High Example 6. Determination of freeze-thaw stability

Starch slurry (5% wb, containing 0.1% sodium azide) of various starches, including waxy cassava (TKWX 1, TKWX 2, TKWX 3 and AM 206-5), waxy corn, waxy rice, waxy potato and normal cassava (Huay Bong 80), was cooked in a boiling water bath for 10 min with continuous stirring. A sample of 1.5 ml cooked paste was transferred to an eppendorf tube of known weight (A), cooled down and the initial weight (B) recorded. The tube was kept at -18°C for 7 days, and then thawed at 25-30°C for 4 hrs for one freeze-thaw cycle. The tube for cycle No. 1 was centrifuged at 10,000 rpm for 10 min, and the expelled water was removed. The weight of the sample was then recorded (C), and % syneresis of starch gel was calculated as below. Other samples were freeze-thawed until the defined cycles were completed, before subjecting them to centrifugation.

% Syneresis = Initial sample weight (B-A) - Final sample weight (C-A) x 100

Initial sample weight (B-A)

Figure 2 illustrates the % syneresis at each freeze-thaw cycle of the various starches. The results suggest that similar to their female parent, AM 206-5, waxy cassava starches from the present invention had the lowest % syneresis, indicating the smallest amount of water was expelled from the cooked pastes. This confirms that waxy cassava starches obtained by the present invention had better freeze-thaw stability than other waxy commercial starches and normal cassava starch. Example 7. Determination of cyanide content in cassava starch

Cyanide content in cassava starches was determined according to the OR-082-TM method recommended by the Ministry of Health, Labor and Welfare,. Japan, Shoku-Ki- Hattsu/Shoku-Kan-Hatsu No. 1 121002; 21 November, 2002. The data are presented in Table 10. Table 10 Cyanide content in starches extracted from various cassava varieties

Note: The cyanide contents were < 50, 50-100 and > 100 ppm for low, moderate and high, respectively.

APPLICABILITY OF THE PRESENT INVENTION The present invention involves the development of waxy starch cassava varieties with better growth performance under the environmental conditions in Thailand. These varieties can provide roots containing waxy cassava starches with improved properties, which can be applied in various industries such as food and beverage, feed, paper and packaging, adhesive and binder, coating, textile and clothing, pharmaceutical, cosmetic, personal care products, hair care products, pet care products and household products.