Carlsberg Res. Commun. Vol. 51, p. 509-513, 1986 A NON-DESTRUCTIVE SCREENING METHOD FOR PROANTHOCYANIDIN-FREE BARLEY MUTANTS by HENRIETTE KRISTENSEN ~ and STEN AASTRUP 2) 1)Department of Physiology, Carlsberg Laboratory and 2}Department of Brewing Chemistry, Carlsberg Research Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby Keywords: Catechin, leucocyanidin, anthocyanin, vanillin-HCl colour reaction Barley grains are embedded in a block of plastic clay with the Carlsberg Seed Fixation System, abraded to expose a small sector of endosperm and surrounding testa. Staining of the proanthocyanidins in the testa is carried out by immersion into a solution of vanillin in 4.5 N-HCI. Kernels lacking proanthocyanidins are selected, washed and grown to mature plants. Using sodium azide mutagenesis 10 proanthocyanidin-free mutants with anthocyanin pigmentation in the vegetative parts of the plants were obtained among 78,450 M3-kernels amounting to a frequency of 0.013%. 1. INTRODUCTION cyanidin (11). A genetic block in the pathway The clear appearance of beer is an important before the branch point, as examplified by quality factor, since many consumers will rank mutants in the genes ant 13, ant 21, ant 17, ant a hazy beer less satisfactory. The most common 22 and ant 18 results in the absence of both forms of haze are precipitates of proteins with anthocyanins and proanthocyanidins. Depend- proanthocyanidins originating from malt or ing on the location of a biochemical mutation hops (5). Therefore, proanthocyanidins have to after the branch point either anthocyanin-free be removed or beer proteins degraded in the plants with normal amounts of catechin and brewing process, in order to achieve a colloidally proanthocyanidins are obtained (ant 1-12, 14- stable bebr. Proanthocyanidin-free barley makes 16), or lines with red anthocyanin pigmentation stabilisation of beer superfluous, as has been lacking catechin and proanthocyanidins can be shown in brewing experiments with the proan- isolated (ant 19). Several hundred mutants, free thocyanidin-free barley mutant ant 13-13 (10). of anthocyanidins and proanthocyanidins, have This has led to extensive breeding programmes been selected by isolating green plants which for proanthocyanidin-free barley varieties (11). lacked anthocyanins in M2-populations in the Proanthocyanidins in the testa of the grains field. From 5 to 25% of such mutants are found and red anthocyanins in the vegetative part of to be proanthocyanidin-free when the kernels the plant are formed by the phenylpropanoid are tested with a vanillin-HC1 solution. This test and flavonoid pathways. As shown in Figure 1 gives a red colour, when catechin or proan- the common precursor of the two classes of thocyanidin are present, and it is carried out on compounds is the (+)-2,3-trans-3,4-cis-leuco- flour from the distal part of the grain, while the Springer-Verlag 0105-1938/86/0051/0509/$01.00 H. KRISTENSEN& S. AASTRUP:Screening for proanthocyanidin-free barley PHENYLALANtNE 1 producer, corn dealer and malster to evaluate a portion of proanthocyanidin-free barley for pu- CINNAMIC ACID rity in a short time. The aim of the present work 1 was to adapt this procedure to a non-destructive ACETYL CoA 4-COUMARIC ACID (SINAPIC ACID) selection ofproanthocyanidin-free mutants and ~ ant21? IFERULICACID) recombinants by carrying out the vanillin test on MALONYL CoA + 4- COUMAROYL CoA.~ (CAFFEIC ACID) sanded kernel surfaces in such a manner that the -- LIGNINS embryo remains undamaged. This allows the selected kernels to be germinated and since a large number of grains can be analysed per day, CHALCONE ... " FLAVANONE an efficient method for selection of proan- ant 22 thocyanidin-free mutants with the vanillin reac- FLAVONE / t tion could be established. DIHYDROFLAVONOL 2. MATERIALS AND METHODS ant 137 FLAVONOL/t ant18 2.1. Grain material LEUCOCYANIDIN For the selection of suitable matrices for ant 1 - 12 / / embedding the kernels and optimal time of 14-1,~ ~L ant 19 staining without damaging the embryo, kernels ANTHOCYAINIDIIN from the varieties Triumph and Galant (a proan- CATECH~N thocyanidin-free mutant in Triumph) were used. For screening of mutagenized material 1 M3-grains harvested on Mz-plants are to be used, PROANTHOCYANIDIN since the proanthocyanidins are synthesized in the maternal tissue (3, 4, 6). To demonstrate the Figure 1. Biosynthesis of proanthocyanidins, an- practical use of the method a total number of thocyanidins and related compounds. The proposed 78,450 M3-kernels of Grit, KM 123 (Research site of action of different ant genes is indicated (9). Institute for Cereal Growing and Breeding, Kromefiz, CSSR) and Catrin were analysed. The mutagen treatment of the grains was carried residual part of the grain containing the embryo out with a l0 3 ~t-sodium azide solution at oH is kept for germination (9). This selection proce- 3.0 for 2 hrs after presoaking of the grains in dure should not lead to mutants which are distilled water at +5 ~ for 15 hrs (9). Prior to blocked in the last step of catechin and proan- harvesting the M,.-populations were examined thocyanidin synthesis i.e. in the portion of the for anthocyanidin-free ME-plants, which were pathway after the branch point, which is specific removed. This increases the probability that the to the biosynthesis of the latter compounds. proanthocyanidin-free mutants found in this Indeed, only very few such mutants have been material most likely contain anthocyanins. found so far. Vanillin staining can be applied on half seeds and thin sections. This permitted the histochem- 2.2. Sample preparation ical location of the proanthocyanidins to the The grains are embedded into a block of clay testa (4). Using the Carlsberg Seed Fixation with the Carlsberg Seed Fixation System (2, 7, 8) System (1) the presence or absence of proan- consisting of matrix pairs, a press and a disc thocyanidins can be determined on sanded sur- sander (Figure 2A and B) as follows: faces of individual barley or malt grains with the 1) Fifty grains are placed into oval-concave vanillin-HCl colour reaction, and 400 kernels depressions of the under-matrix with the aid of can be analysed destructively in a period of a few the over-matrix containing oval holes (Figure minutes (1), permitting the plant breeder, seed 2C). 510 Carlsberg Res. Commun. Vol. 51, p. 509-513, 1986 H. KRISTENSEN& S. AAS'I'RUP:Screening for proanthocyanidin-free barley Figure 2. Carlsberg Seed Fixation System, A: Matrix pairs and press. B: Disc sander. C: Over-matrix and under-matrix containing grains. D: Clay block with grains. E: Clay block with lightly sanded grains. F: Clay block with vanillin-HCl stained grains. G: Proanthocyanidin-containinggrain stained with vanillin-HCl. H: Proanthocyanidin-free grain stained with vanillin-HC1. Carlsberg Res. Commun. Vol. 51, p. 509-513, 1986 511 H. KRISTENSEN& S. AASTRUP:Screening for proanthocyanidin-free barley 2) The grains are fixed in a block of clay, (Filia Tabel I. Screening for proanthocyanidin-free M3- a/s, J~egersborgvej 64, DK-2800 Lyngby, Den- grains in three barley lines, treated with the mutagen mark) by means of the press (Figure 2D). sodium azide. 3) A small outer part of each grain is removed Grit KM 123 Catrin with the disc sander and this uncovers a sector of the white endosperm (Figure 2E). Screened grains 52200 20000 6250 Selected grains without distinct 117 49 10 2.3. Staining red staining The proanthocyanidins are rendered visible Germinated grains 67 26 8 by staining the sanded grains with a freshly Survivors 53 21 7 prepared solution of 1% vanillin in HC1. The solution is stable for 3-4 hours. A red colour Proanthocyanidin- 7 4 2 develops in the testa of proanthocyanidin-con- free plants taining grains (Figure 2F and G), while proan- Proanthocyanidin- thocyanidin-free grains remain colourless (Fig- free plants con- 5 3 2 ure 2F and H). taining anthocyanin 2.4. Selection procedure Screening for the proanthocyanidin-free mu- tants is carried out with the naked eye or a 3.2. Application magnifying glass. The optimal position for look- The number of proanthocyanidin-free mu- ing at the plates is from an oblique angle. tants obtained with this method are listed in Grains lacking a red ring around the en- Table I. In the three lines the frequency of dosperm are taken out and instantly placed in a apparently proanthocyanidin-free grains was glass of pure water. To rinse out the residual acid between 0.16% and 0.24%. Germination of the the selected grains are placed in running water selected grains varied from 57% in Grit, to 53% (e.g. in a small sieve) over night. in KM 123, and 80% in Catrin. The reason for The following day, the seeds are sown in this low germination rate in the progeny of the vermiculite. A couple of days after germination, mutagenized grains as compared to the untreat- they are transfered to soil. ed Galant kernels is possibly due to segregating mutant kernels with decreased viability. The majority of the plants that withered and died 3. RESULTS AND DISCUSSION after germination were albina mutants. 3.1. Development of the method Whether or not the plant had anthocyanin Grains from the lines Triumph and Galant pigmentation was registered during growth. Af- were used in developing the procedure. Various ter harvesting, each plant was checked for proan- combinations of acidity of the solution and the thocyanidin content with the vanillin test car- times of immersion of the grains were tested to tied out on one fourth of each single grain (9). find an acceptable combination ofcolour inten- This test showed that 13 of the selected and sity and germination ability. The colour devel- surviving plants were proanthocyanidin-free opment for Triumph was satisfactory after treat- mutants, which is commensurate with 16%.