C.L. Rife1, C. Blaker1, and G. Sidlauskas2


1Kansas State University, 2004 Throckmorton Hall, Manhattan, KS 66506, USA.

2Lithuanian Institute of Agriculture, Dotnuva-Akademija, 5051 Kedainiai, Lithuania.



Production of rapeseed for canola oil has increased over the past few decades.  Areas where rapeseed had not been grown previously are now being considered for production.  Some potential production areas have soils with low pH and toxic levels of aluminum (Al).  Cultivars of several crops have been developed that are more tolerant to Al and are capable of higher yields with reduced levels of lime.  A screening procedure was based on procedures developed for other crops.  Six days after seeds were imbibed, seedlings were suspended in a liquid medium containing high levels of Al.  On day 7, roots of the seedlings were placed in a hematoxylin solution to identify damage from Al. Root tips were scored from 0 (no staining, no damage) to 4 (dark staining, severe damage).  Available released cultivars and the Plant Introduction collection (total of 546 lines) were screened for reaction to Al.  Most of the recently developed cultivars were rated susceptible to highly susceptible for Al damage, but several PI lines with tolerance to Al were identified.


KEYWORDS:  Plant Introduction Collection, Soil pH




Some types of rapeseed produce the edible oil canola, which is known for its many health benefits.  In 1997, approximately 730,000 acres of canola were grown in the U.S. and the acreage increased to 1.13 million acres in 1998 (Anonymous, 1997).  Although domestic production is increasing, two-thirds of the U.S. consumption still is supplied from imports.  Initial attempts to produce canola in the Great Plains were not successful.  This was due partly to a lack of adapted cultivars and partly to a lack of knowledge about management requirements for this area.  Over the past 5 years, progress has been made in achieving yields that are economically competitive with those of other crops grown in the region (Rife, 1998).  Some potential production areas have soils with low pH and toxic levels of  aluminum (Al).  Southeast and south-central Kansas, western Missouri, and north-central Oklahoma possess areas with low pH soils and experience Al toxicity problems.  Aluminum is a component of all soil minerals and is found in the forms of layered silicates, oxide minerals, and soluble acids (Thomas and Margrove, 1984).  As soil becomes more acidic, aluminum solubilizes into the soil solution and is available for root absorption.  Plant species and cultivar genotypes differ in their responses to acid soils (Foy, 1976; Neenan 1960).  Barley is very sensitive to acid soils, red clover and wheat are sensitive, and oats and rye are tolerant (Russel, 1973).  Canola's tolerance to acid soils is very similar to that of wheat -- soil pH below 5.5 reduces yields (Thomas, 1984).  Genetic variability for Al tolerance is available in wheat, and cultivars have been developed that tolerate Al soils with little yield reduction.  Wheat cultivars appearing most tolerant were developed in Brazil, Ohio, North Carolina, Georgia, and Kansas -- regions where acid soils are present (Foy, et al.,  1965). 


Screening procedures to determine genetic tolerance must be quick, reliable, and capable of handling large numbers of plants (Foy, 1976).  Current available methods differ, but all include exposing actively growing plants to Al.  With wheat, this procedure correlated extremely well with field results (Unruh, 1989).  The objectives of this study were to develop a screening procedure and evaluate genetic variability in tolerance to Al toxicity of rapeseed at the seedling stage.



Three trays with 40 cells each (15 mm x 40 mm) were used to germinate seeds.  Six seeds each of 120 lines were placed in a cell on blotter paper moistened with a 0.5 g l-1 Terrocoat solution.  To optimize uniform germination, trays were placed at room temperature for 12 hr, incubated at 10 Co for 72 hr, and then returned to room temperature for an additional 36 hr, all in darkness.  Seedlings were placed on plastic screens (2.5 mm mesh with grids painted to allow identification of individual seedlings) and their roots were threaded through the screens. The screen were in turn floated in full- strength Hoaglands solution (Hoagland and Arnon, 1950) buffered to pH 4.0 for 24 hr under continuous light and aeration.  The light was provided by two fluorescent grow lights suspended 34 cm above the seedlings.  The nutrient solution then was discarded and replaced with fresh nutrient solution containing Al (AlCl3 6H2O) and buffered to pH 4.0.  Seedling were kept in this solution for 17 hr under continuous light and aeration.  A series of experiments was conducted to determine the most appropriate concentration for germplasm screening.  Three concentrations of Al (4, 6, and 8 mM) were tested and 8 mM was used on subsequent screenings.  After the Al treatment, the seedlings were washed in aerated distilled-deionized water for 1 hr to remove any Al remaining on the root surface.  A stain was prepared using 2 g l-1 hematoxylin and 0.2 g l-1 NaIO3  and was mixed overnight with a magnetic stirrer.  The screens containing the seedlings were placed in the hematoxylin solution for 15 min.  The stained roots were rinsed in flowing distilled-deionized water, and then screens of seedlings were placed in aerated distilled-deionized water under continuous light for 24 hr.  The concentration experiments screened 36 rapeseed cultivars and experimental lines as well as 3 wheat cultivars used as checks.  '2163' wheat is known to be tolerant to Al, 'Karl 92' is intermediate, and 'TAM 107' is susceptible to Al toxicity.  All available Plant Introduction (PI) lines were also screened (510 lines, most are not canola quality).


Each seedling was rated visually and an average score was determined for each line.  The rating was based on the degree of staining at the root tip, which was evaluated against a white background.  The scoring system was as follows: 0 = no staining; 1 = slight staining with clear regrowth; 2 = increased staining with stained regrowth;  3 = dark staining with stained regrowth;  4 = dark staining with no regrowth. 




Differences in the amount of damage caused by Al treatment to the developing root tip were detected.  Trends indicated that as the Al concentration was increased from 4 mM to 8 mM Al, the amount of damage to the seedling root tip also increased (Table 1).  At these concentrations, however, significant differences were not observed.  Significant differences were detected between lines within concentration treatments.  The wheat check 2163 (tolerant of aluminum and recommended for soils with low pH) had a significantly lower score than any rapeseed line shown in Table 1 (modern cultivars and experiment lines).  Another wheat check, TAM 107 (highly susceptible to acid soils), was not significantly different from the rapeseed lines with the highest scores. 


Of the 546 lines evaluated, half received scores of 1.75 or lower and only 3 of these were included in the concentration experiments (Figure 1).  Twenty-four of  the PI lines had scores of 0.00.  The mean score of the lines tested from the PI collection (1.79) was less than the mean score of the 36 available canola cultivars and advanced experimental lines (2.97).  Several factors could be involved with the differences between the groups.  Differences in response also were detected for the area of origin (Table 2).  Modern winter canola lines common in the United States may be originating in areas that historically have developed rapeseed lines without good tolerance to AL

Table 1.  Aluminum Screening Results with 36 Rapeseed  Lines and 3 Wheat Cultivars at 3

Aluminum Concentrations.  


Lines                                                     4 mM        6 mM        8 mM


2163 (wheat)                                         0.00           0.00           0.00

WW1089                                               2.50           1.25           1.33

ID.WR.465.2.4                                     2.00           2.00           1.67

MLCH 50                                             2.50           3.50           1.67

Aspen                                                   1.50           2.00           2.00

ID.92.WC.3.13.4                                  2.50           3.50           2.00

Ceres                                                     2.00           3.50           2.00

Erika                                                      2.75           3.25           2.00

MLCH 34                                             1.75           0.50           2.00

KS3580                                                 1.50           2.50           2.33

ARC-12L-3                                           2.00           3.50           2.33

Cathy                                                    2.75           2.75           2.33

7-88A                                                    1.50           1.50           2.33

Jetton                                                    2.00           2.00           2.50

ID.92.SW.76.75                                    1.50           2.50           2.50

Karl 92 (wheat)                                     3.00           3.00           2.67

Accord                                                  2.00           2.00           2.67

Bingo                                                     3.50           3.50           2.67

Selkirk                                                   1.25           1.25           3.00

KS1701                                                 3.00           3.00           3.00

UGA488                                               2.00           4.00           3.00

A112                                                     2.00           1.50           3.00

Glacier                                                   1.75           2.25           3.00

TAM 107 (wheat)                                3.00           3.00           3.00

ID.92.WC.2.24.5                                  2.00           2.00           3.33

KS3579                                                 2.50           3.00           3.50

Liborius                                                 3.25           2.25           3.50

Casino                                                   3.00           3.50           3.67

D931                                                     2.50           3.00           3.67

Plainsman                                              3.00           4.00           4.00

ARC-7L-3                                             2.50           3.50           4.00

Bridger                                                  3.00           3.00           4.00

ARC-59L-4                                           1.50           3.00           4.00

Winfield                                                2.50           3.50           4.00

MO503-1                                              3.00           3.50           4.00

KS3203                                                 2.50           1.50           4.00

Falcon                                                   4.00           3.50           4.00

DCH 29                                                                  2.50           2.50                4.00

K747D                                                  4.00           3.50           4.00


  Means                                                 2.31           2.59           2.83

  LSD (Treat: 0.05)                               ------------ NS ------------ 

  LSD (Line w/ Treat)                           ----------- 1.23  ----------- 

  CV (%)                                                ----------- 29.54 ----------- 


* Rated on the basis of hematoxylin staining (0 = no staining, no damage to 4 = dark staining, severe damage).  Presented in order of rating with 8 mM Al.


toxicity.  However, when the 36 canola lines were divided by country of origin, in all cases, their mean score was higher than the mean score of the older rapeseed lines from that country in the PI

collection.  Management practices also have changed over the last several decades.  It is now common for soil pH problems to be corrected with soil amendments, especially in research plots.  When the soil in a breeding nursery is naturally neutral or artificially corrected to a neutral pH, progress in selecting for genotypes more tolerant to toxic levels of Al will not be made.  The genes associated with erucic acid or glucosinolates also may contribute to the Al response or may be linked with the genes that are responsible.


Figure 1. Frequencies of aluminum scores within four different ranges.  Lower                     scores are associated with tolerance to aluminum, and higher scores are                     associated with susceptibility to aluminum.


Table 2.  Aluminum Damage Scores of Rapeseed Lines Developed in Different Countries.           


Country                       No. of      Mean Damage

of Origin                      Lines                    Score


India                                     1                      0.80

Nepal                                    1                      1.00

Poland                                21                      1.18

Sweden                              12                      1.25

Canada                                 6                      1.39

Norway                                1                      1.40

Japan                                  55                      1.44

Pakistan                               3                      1.45

USA                                     4                      1.59

New Zealand                       8                      1.61

Germany                            50                      1.71

France                                26                      1.72

Bangladesh                         5                      1.81

Taiwan                                 4                      1.83

Netherlands                        1                      2.00

Hungry                                5                      2.02

Russia                                  4                      2.05

Czech                                   3                      2.17

Romania                               1                      2.33

China                                    9                      2.34

Yugoslavia                          1                      2.60

Algeria                                 1                      2.67

Turkey                                 3                      2.73


* Rated on the basis of hematoxylin staining (0 = no staining, no damage to 4 = dark staining, severe damage).

Yield studies to complement these results currently are underway in the greenhouse and are planned  for the field.   Further field screening of identified experimental lines will help in the development

of cultivars more tolerant to Al toxicity.  Many areas of Kansas and the rest of the Great Plains are not well suited to summer annual crops and have pockets where soil pH is dangerously low.  Liming is not always an economical option for many of these areas, and the development of wheat cultivars adapted to the Great Plains that are tolerant to high level of Al has allowed many of these marginal farms to become more profitable.  The addition of adapted canola cultivars with increased levels of Al tolerance will give producers another cropping option.




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