1144
Specialia
t h e w h i t e eye s t o c k a n d cytological e x a m i n a t i o n of t h e p r o g e n y (larvae) w a s c a r r i e d o u t to assess w h i c h m a l e s were i n v e r s i o n h e t e r o z y g o t e s . O n l y m a l e s were used d u r ing t h i s step to rule o u t crossing o v e r w h i c h w o u l d confuse t h e c h r o m o s o m e - m a r k e r r e l a t i o n s h i p . I n t h e following g e n e r a t i o n (G3), t w o s i t u a t i o n s are possible d e p e n d i n g o n t h e l o c a t i o n of t h e eye color. I f it is located on c h r o m o some 3, t h e n all red eye flies will b e i n v e r s i o n h e t e r o z y gous a n d all w h i t e eye flies are n o r m a l . If t h e m a r k e r gene is on a n y o t h e r c h r o m o s o m e , h a l f of b o t h t h e r e d eye a n d w h i t e eye flies will be i n v e r s i o n h e t e r o z y g o u s a n d h a l f will b e n o r m a l . Males b e i n g m u c h easier to analyse, we c h e c k e d in G3, 18 w+w (red) m a l e s w h i c h were all i n v e r sion h e t e r p z y g o t e s ; a n d 19 ww males w h i c h were all normal. T h i s is a s t r o n g i n d i c a t i o n for t h e linkage of t h e eye color g e n e w i t h c h r o m o s o m e 3. w+w males were a g a i n t e s t crossed w i t h n o r m a l ww females in o r d e r t o r e p e a t t h e a n a l y s i s in t h e n e x t g e n e r a t i o n G 4. A g a i n all ww m a l e s (33) were n o r m a l , a n d all w+w (red) m a l e s (7) i n v e r s i o n heterozygous. To see w h e t h e r r e c o m b i n a t i o n b e t w e e n t h e i n v e r s i o n a n d t h e m a r k e r gene occurs, we h a v e a n a l y z e d t h e a d u l t m a l e G 4 p r o g e n y of w+w females. I t a p p e a r e d t h a t b o t h w+w a n d ww m a l e s c a n b e i n v e r s i o n h e t e r o z y g o u s or normal. A r e c o m b i n a t i o n of 4 0 . 2 % was f o u n d ( N = 1 1 2 ) . A i h g h incidence of d o u b l e crossovers w i t h i n t h e i n v e r s i o n
EXPERIENTIA 32/9
b e i n g unlikely, t h i s suggests t h a t t h e eye colour locus is s i t u a t e d d i s t a l l y o n t h e long a r m of c h r o m o s o m e 3. L i n k a g e b e t w e e n t h e i n v e r s i o n a n d t h e red eye m a r k e r c a n be m a i n t a i n e d b y r o u t i n e l y r e p e a t i n g t h e G a a n d G 4 crossings w i t h o u t a n y f u r t h e r a n a l y s i s of k a r y o t y p e s or fertility. T h i s l i n k a g e e n a b l e s sibcrosses t o b e m a d e b e t w e e n inv e r s i o n h e t e r o z y g o u s m a l e s a n d females to o b t a i n inv e r s i o n h o m o z y g o t e s j u s t b y c o m b i n i n g t h e r e d eye p a r t n e r s f r o m r e d eye fathers. T h e s e h o m o z y g o t e s , if viable, c a n b e used for genetic i n s e c t control. A n o t h e r a p p l i c a t i o n for genetic i n s e c t c o n t r o l purposes, as suggested b y PARKER I, is t h e p o s s i b i l i t y of isolating compound chromosomes after irradiating inversion heretozygotes. This c a n be c a r r i e d o u t w i t h t h i s i n v e r s i o n b y i r r a d i a t i n g w+w, In~+ m a l e s a n d s u b s e q u e n t l y crossing t h e m w i t h t h e w+w, In~+ females. A f t e r t h e i n d u c t i o n b y i r r a d i a t i o n of c r o s s i n g o v e r in t h e male, d u p l i c a t i o n / d e ficiency g a m e t e s m a y o c c u r w h i c h are c o m p l e m e n t e d in a few c o m b i n a t i o n s a n d give a c o m p o u n d k a r y o t y p e . Comp o u n d s t r a i n s h a v e some i n t r a - s t e r i l i t y , a n d c o m p l e t e s t e r i l i t y a f t e r crossing w i t h t h e wild t y p e in t h e field.
D. R. PARKER, Biological Control, The University of Texas 5914), 113 (1959).
Chloral Hydrate Induced Haploidization in Aspergillus nidulans M. SINGH a n d U. SIN~IA
DeparLment o/ Botany, University o/Delhi, Delhi-110007 (India), 26 January 1976. Summary. T h i s is t h e first r e p o r t of i n d u c t i o n of h a p l o i d i z a t i o n in Aspergillus nidulans b y chloral h y d r a t e , w h i c h is a n efficient p o l y p l o i d i z i n g a g e n t for h i g h e r p l a n t s a n d a p s y c h o t r o p i c d r u g for m a n . A n e w p r o c e d u r e h a s b e e n d e s c r i b e d to isolate h a p l o i d s f r o m diploids w i t h a v e r y h i g h f r e q u e n c y , as c o m p a r e d t o p - f l u o r o p h e n y l a l a n i n e , w h i c h is generally used for t h i s p u r p o s e . Chloral h y d r a t e is w i d e l y used as a p s y c h o t r o p i c d r u g a n d as a n a n a e s t h e t i c . I t h a s b e e n s h o w n to interfere w i t h t h e f o r m a t i o n of m i t o t i c s p i n d l e in p l a n t s 1, in g r a s s h o p p e r s p e r m a t o c y t e s 2, in Pleurodeles waltlii a a n d in Aspergillus nidulans4. W e wish to r e p o r t here a n e w a n d i n t e r e s t i n g p r o p e r t y of chloral h y d r a t e in i n d u c i n g h a p l o i d i z a t i o n in Aspergillus nidulans diploids. H e t e r o z y g o u s diploids, w h i c h c a n b e easily isolated in t h i s f u n g u s ~, give rise t o n e w diploids a n d h a p l o i d s s p o n t a n e o u s l y ~, t h o u g h a t a v e r y low f r e q u e n c y . T h e h a p l o i d s e g r e g a n t s c a n be v i s u a l l y d e t e c t e d in a diploid w h i c h is h e t e r o z y g o u s for c o n i d i a l colour m a r k e r s . Chloral h y d r a t e was i n c o r p o r a t e d in t h e s t a n d a r d c o m p l e t e m e d i u m ~ t o v a r i o u s final c o n c e n t r a t i o n s r a n g ing f r o m 0.001 M t o 0.04 M , a n d c o n i d i a f r o m diploid s t r a i n s h e t e r o z y g o u s for 2 c o l o u r m a r k e r s as well as a n u m b e r of n u t r i t i o n a l m a r k e r s a n d t h e c o r r e s p o n d i n g h a p l o i d s were p l a t e d o n t h e surface. V i a b l e c o u n t s were d r a s t i c a l l y affected b y i n c r e a s i n g c o n c e n t r a t i o n s of c h l o r a l h y d r a t e , so m u c h so t h a t a t 0.02 M , o n l y 2~o of c o n i d i a could f o r m colonies. T h e r e was no d i f f e r e n t i a l effect o n h a p l o i d s a n d diploids. H o w e v e r , w h e n a t h i c k i n o c u l u m of c o n i d i a was s t r e a k e d , r a t h e r thai1 s t a b b e d or p l a t e d , t h e g r o w t h was a little b e t t e r . A t a c o n c e n t r a t i o n of 0.02 M c h l o r a l h y d r a t e , s t r e a k s of diploid s t r a i n s p r o d u c e d well
Fig. 1. A streak of a heterozygous diploid showing small sectors on complete medium + 0.02 M chloral hydrate after 12 days of incubation.
15.9. 1976
1145
Specialia
Mitotic segregation induced by chloral hydrate in different diploids Diploid used
No. of streaks analyzed
No. of segregants analyzed Yellow
White
Green
Total
No. of haploid segregants
No. of diploid segregants
No. of classes of haploid segregants
On chloral hydrate alone
riboA1, biA1; 1paX71
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8
9
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17
17
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13
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21
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MSE~ On chloral hydrate and then transferred to complete medium
riboA 1, biA 1; ]paXTO MSE
riboA 7, biA 1; /pax72 MSE MSE, Master Strain 'E 's.
c o n i d i a t i n g a n d fast g r o w i n g sectors a f t e r a b o u t 10-12 d a y s of i n c u b a t i o n . E a c h s t r e a k gave rise t o a b o u t 15 d i s t i n c t sectors, w h i c h w h e n isolated a n d t e s t e d s e p a r a t e l y for conidial d i a m e t e r a n d b i o c h e m i c a l r e q u i r e m e n t s , were f o u n d to be h a p l o i d s a n d did n o t segregate f u r t h e r on a m e d i u m c o n t a i n i n g p - f l u o r o p h e n y l a l a n i n e (FPA). A b o u t 85% of t h e m were d i f f e r e n t f r o m one a n o t h e r , i n d i c a t i n g t h a t t h e clonal factor was g r e a t l y d i m i n i s h e d . The g r o w t h of t h e diploid in t h e s t r e a k is n o t d e t e c t a b l e visually, a n d all t h e s e g r e g a n t s p r o d u c e d are h a p l o i d s i n d i c a t i n g a selective effect of chloral h y d r a t e a g a i n s t diploids a n d disomics. E v e n t h e h a p l o i d s do n o t grow v e r y well a n d a p p e a r a f t e r 8-10 d a y s as smaI1 sectors (Figure 1). If t h e s t r e a k is t r a n s f e r r e d to c o m p l e t e m e d i u m a f t e r 5 d a y s on chloral h y d r a t e , t h e sectors grow o u t v i g o r o u s l y a n d s p o r u l a t e n o r m a l l y w i t h i n 2 d a y s of t h e t r a n s f e r (Figure 2), t h e r e b y facilitating isolation. A l t h o u g h all g r o w t h a p p e a r s b r o w n ish grey on chloral h y d r a t e , on c o m p l e t e m e d i u m sectors w i t h d i f f e r e n t colours are e x t r e m e l y p r o m i n e n t because of
Fig. 2. The diploid was first streaked on complete medium + 0.02 M chloral hydrate, incubated for 5 days and then transferred to complete medium. After 2-3 days of the transfer, fast growing and profusely conidiating sectors are visible.
profuse conidiation. Out of 36 isolates picked up from s t r e a k s first g r o w n on chloral h y d r a t e a n d t h e n t r a n s ferred to c o m p l e t e m e d i u m , 35 t u r n e d o u t to be haploids a n d only one was a diploid (Table), i n d i c a t i n g t h e r e b y t h a t , d u r i n g 5 d a y s on chloral h y d r a t e , t h e r e is a l m o s t c o m p l e t e h a p l o i d i z a t i o n , following w h i c h t h e h a p l o i d sect o r s grow o u t of t h e streak. Using chloral h y d r a t e as t h e h a p l o i d i z i n g agent, 2 n e w l y i s o l a t e d F P A - r e s i s t a n t m u t a n t s (riboA 1, biA I; [flaX70 a n d riboA 1, biA L"/paX71) were assigned to t h e linkage g r o u p VI, a n d t h e t h i r d n e w isolate (riboA 1, biA 1;/paX72) was assigned to t h e linkage g r o u p i. These a s s i g n m e n t s were c o n f i r m e d b y using F P A as t h e h a p l o i d i z i n g a g e n t 9. S e g r e g a t i o n of all o t h e r m a r k e r s was as e x p e c t e d on t h e basis of t h e i r linkage g r o u p locations. T h e diploid b e t w e e n / p a X 7 0 a n d M S E gave o u t o n l y g r e e n sectors w h e n F P A was used as t h e h a p l o i d i z i n g agent, b u t on choral h y d r a t e well growing a n d p r o f u s e l y c o n i d i a t i n g yellow, w h i t e a n d g r e e n sectors could easily be o b t a i n e d . The above-mentioned chemical and the procedure make t h e i n d u c t i o n a n d isolation of h a p l o i d s f r o m a diploid less t i m e c o n s u m i n g a n d v e r y easy, t h u s facilitating t h e entire p r o c e d u r e of genetic m a p p i n g b a s e d on m i t o t i c segregation. Also, chloral h y d r a t e is m u c h c h e a p e r t h a n p-fluorop h e n y l a l a n i n e . I t is u n i q u e because of its dual f u n c t i o n i n d u c t i o n of c-mitosis a n d p o l y p l o i d y in higher p l a n t s and h a p l o i d i z a t i o n in Aspergillus. To our knowledge, t h e r e is no r e p o r t of efficient i n d u c t i o n of h a p l o i d i z a t i o n b y a n y of t h e polyploidizing a g e n t s in a n y organism. Thus, chloral h y d r a t e d e s e r v e s m o r e a t t e n t i o n as a p o t e n t i a l h a p l o i d i z i n g a g e n t for p l a n t a n d a n i m a l ceils. Since it is a w i d e l y used drug, such i n v e s t i g a t i o n s would a d d to our k n o w l e d g e r e g a r d i n g its effects on h u m a n beings.
1 A. LEWAN,Hereditas 2d, 471 (1938). 2 H. Ris, Biol. Bull. 96, 90 (1949). 3 p. SENTEIN, Chromosoma dS, 215 (1974). 4 B. M~RCER and N. R. MORRIS, J. gen. Mierobiol. 88, 197 (1975). 5 j. A. ROPER, Experientia 8, 14 (1952). G. PONTECORVO,A. Rev. Microbiol. 70, 393 (1956). U. SINHA, Genetics 62, 495 (1969). 8 K. S. MCCULLYand E. FORBES, Genet. Res. 6, 352 (1965). 9 p. LHOAS, Nature, Lond. 790, 794 (1961).
Specialia
t h e w h i t e eye s t o c k a n d cytological e x a m i n a t i o n of t h e p r o g e n y (larvae) w a s c a r r i e d o u t to assess w h i c h m a l e s were i n v e r s i o n h e t e r o z y g o t e s . O n l y m a l e s were used d u r ing t h i s step to rule o u t crossing o v e r w h i c h w o u l d confuse t h e c h r o m o s o m e - m a r k e r r e l a t i o n s h i p . I n t h e following g e n e r a t i o n (G3), t w o s i t u a t i o n s are possible d e p e n d i n g o n t h e l o c a t i o n of t h e eye color. I f it is located on c h r o m o some 3, t h e n all red eye flies will b e i n v e r s i o n h e t e r o z y gous a n d all w h i t e eye flies are n o r m a l . If t h e m a r k e r gene is on a n y o t h e r c h r o m o s o m e , h a l f of b o t h t h e r e d eye a n d w h i t e eye flies will be i n v e r s i o n h e t e r o z y g o u s a n d h a l f will b e n o r m a l . Males b e i n g m u c h easier to analyse, we c h e c k e d in G3, 18 w+w (red) m a l e s w h i c h were all i n v e r sion h e t e r p z y g o t e s ; a n d 19 ww males w h i c h were all normal. T h i s is a s t r o n g i n d i c a t i o n for t h e linkage of t h e eye color g e n e w i t h c h r o m o s o m e 3. w+w males were a g a i n t e s t crossed w i t h n o r m a l ww females in o r d e r t o r e p e a t t h e a n a l y s i s in t h e n e x t g e n e r a t i o n G 4. A g a i n all ww m a l e s (33) were n o r m a l , a n d all w+w (red) m a l e s (7) i n v e r s i o n heterozygous. To see w h e t h e r r e c o m b i n a t i o n b e t w e e n t h e i n v e r s i o n a n d t h e m a r k e r gene occurs, we h a v e a n a l y z e d t h e a d u l t m a l e G 4 p r o g e n y of w+w females. I t a p p e a r e d t h a t b o t h w+w a n d ww m a l e s c a n b e i n v e r s i o n h e t e r o z y g o u s or normal. A r e c o m b i n a t i o n of 4 0 . 2 % was f o u n d ( N = 1 1 2 ) . A i h g h incidence of d o u b l e crossovers w i t h i n t h e i n v e r s i o n
EXPERIENTIA 32/9
b e i n g unlikely, t h i s suggests t h a t t h e eye colour locus is s i t u a t e d d i s t a l l y o n t h e long a r m of c h r o m o s o m e 3. L i n k a g e b e t w e e n t h e i n v e r s i o n a n d t h e red eye m a r k e r c a n be m a i n t a i n e d b y r o u t i n e l y r e p e a t i n g t h e G a a n d G 4 crossings w i t h o u t a n y f u r t h e r a n a l y s i s of k a r y o t y p e s or fertility. T h i s l i n k a g e e n a b l e s sibcrosses t o b e m a d e b e t w e e n inv e r s i o n h e t e r o z y g o u s m a l e s a n d females to o b t a i n inv e r s i o n h o m o z y g o t e s j u s t b y c o m b i n i n g t h e r e d eye p a r t n e r s f r o m r e d eye fathers. T h e s e h o m o z y g o t e s , if viable, c a n b e used for genetic i n s e c t control. A n o t h e r a p p l i c a t i o n for genetic i n s e c t c o n t r o l purposes, as suggested b y PARKER I, is t h e p o s s i b i l i t y of isolating compound chromosomes after irradiating inversion heretozygotes. This c a n be c a r r i e d o u t w i t h t h i s i n v e r s i o n b y i r r a d i a t i n g w+w, In~+ m a l e s a n d s u b s e q u e n t l y crossing t h e m w i t h t h e w+w, In~+ females. A f t e r t h e i n d u c t i o n b y i r r a d i a t i o n of c r o s s i n g o v e r in t h e male, d u p l i c a t i o n / d e ficiency g a m e t e s m a y o c c u r w h i c h are c o m p l e m e n t e d in a few c o m b i n a t i o n s a n d give a c o m p o u n d k a r y o t y p e . Comp o u n d s t r a i n s h a v e some i n t r a - s t e r i l i t y , a n d c o m p l e t e s t e r i l i t y a f t e r crossing w i t h t h e wild t y p e in t h e field.
D. R. PARKER, Biological Control, The University of Texas 5914), 113 (1959).
Chloral Hydrate Induced Haploidization in Aspergillus nidulans M. SINGH a n d U. SIN~IA
DeparLment o/ Botany, University o/Delhi, Delhi-110007 (India), 26 January 1976. Summary. T h i s is t h e first r e p o r t of i n d u c t i o n of h a p l o i d i z a t i o n in Aspergillus nidulans b y chloral h y d r a t e , w h i c h is a n efficient p o l y p l o i d i z i n g a g e n t for h i g h e r p l a n t s a n d a p s y c h o t r o p i c d r u g for m a n . A n e w p r o c e d u r e h a s b e e n d e s c r i b e d to isolate h a p l o i d s f r o m diploids w i t h a v e r y h i g h f r e q u e n c y , as c o m p a r e d t o p - f l u o r o p h e n y l a l a n i n e , w h i c h is generally used for t h i s p u r p o s e . Chloral h y d r a t e is w i d e l y used as a p s y c h o t r o p i c d r u g a n d as a n a n a e s t h e t i c . I t h a s b e e n s h o w n to interfere w i t h t h e f o r m a t i o n of m i t o t i c s p i n d l e in p l a n t s 1, in g r a s s h o p p e r s p e r m a t o c y t e s 2, in Pleurodeles waltlii a a n d in Aspergillus nidulans4. W e wish to r e p o r t here a n e w a n d i n t e r e s t i n g p r o p e r t y of chloral h y d r a t e in i n d u c i n g h a p l o i d i z a t i o n in Aspergillus nidulans diploids. H e t e r o z y g o u s diploids, w h i c h c a n b e easily isolated in t h i s f u n g u s ~, give rise t o n e w diploids a n d h a p l o i d s s p o n t a n e o u s l y ~, t h o u g h a t a v e r y low f r e q u e n c y . T h e h a p l o i d s e g r e g a n t s c a n be v i s u a l l y d e t e c t e d in a diploid w h i c h is h e t e r o z y g o u s for c o n i d i a l colour m a r k e r s . Chloral h y d r a t e was i n c o r p o r a t e d in t h e s t a n d a r d c o m p l e t e m e d i u m ~ t o v a r i o u s final c o n c e n t r a t i o n s r a n g ing f r o m 0.001 M t o 0.04 M , a n d c o n i d i a f r o m diploid s t r a i n s h e t e r o z y g o u s for 2 c o l o u r m a r k e r s as well as a n u m b e r of n u t r i t i o n a l m a r k e r s a n d t h e c o r r e s p o n d i n g h a p l o i d s were p l a t e d o n t h e surface. V i a b l e c o u n t s were d r a s t i c a l l y affected b y i n c r e a s i n g c o n c e n t r a t i o n s of c h l o r a l h y d r a t e , so m u c h so t h a t a t 0.02 M , o n l y 2~o of c o n i d i a could f o r m colonies. T h e r e was no d i f f e r e n t i a l effect o n h a p l o i d s a n d diploids. H o w e v e r , w h e n a t h i c k i n o c u l u m of c o n i d i a was s t r e a k e d , r a t h e r thai1 s t a b b e d or p l a t e d , t h e g r o w t h was a little b e t t e r . A t a c o n c e n t r a t i o n of 0.02 M c h l o r a l h y d r a t e , s t r e a k s of diploid s t r a i n s p r o d u c e d well
Fig. 1. A streak of a heterozygous diploid showing small sectors on complete medium + 0.02 M chloral hydrate after 12 days of incubation.
15.9. 1976
1145
Specialia
Mitotic segregation induced by chloral hydrate in different diploids Diploid used
No. of streaks analyzed
No. of segregants analyzed Yellow
White
Green
Total
No. of haploid segregants
No. of diploid segregants
No. of classes of haploid segregants
On chloral hydrate alone
riboA1, biA1; 1paX71
1
8
9
0
17
17
0
13
2
13
7
1
21
21
0
15
2
10
4
2
16
15
1
12
MSE~ On chloral hydrate and then transferred to complete medium
riboA 1, biA 1; ]paXTO MSE
riboA 7, biA 1; /pax72 MSE MSE, Master Strain 'E 's.
c o n i d i a t i n g a n d fast g r o w i n g sectors a f t e r a b o u t 10-12 d a y s of i n c u b a t i o n . E a c h s t r e a k gave rise t o a b o u t 15 d i s t i n c t sectors, w h i c h w h e n isolated a n d t e s t e d s e p a r a t e l y for conidial d i a m e t e r a n d b i o c h e m i c a l r e q u i r e m e n t s , were f o u n d to be h a p l o i d s a n d did n o t segregate f u r t h e r on a m e d i u m c o n t a i n i n g p - f l u o r o p h e n y l a l a n i n e (FPA). A b o u t 85% of t h e m were d i f f e r e n t f r o m one a n o t h e r , i n d i c a t i n g t h a t t h e clonal factor was g r e a t l y d i m i n i s h e d . The g r o w t h of t h e diploid in t h e s t r e a k is n o t d e t e c t a b l e visually, a n d all t h e s e g r e g a n t s p r o d u c e d are h a p l o i d s i n d i c a t i n g a selective effect of chloral h y d r a t e a g a i n s t diploids a n d disomics. E v e n t h e h a p l o i d s do n o t grow v e r y well a n d a p p e a r a f t e r 8-10 d a y s as smaI1 sectors (Figure 1). If t h e s t r e a k is t r a n s f e r r e d to c o m p l e t e m e d i u m a f t e r 5 d a y s on chloral h y d r a t e , t h e sectors grow o u t v i g o r o u s l y a n d s p o r u l a t e n o r m a l l y w i t h i n 2 d a y s of t h e t r a n s f e r (Figure 2), t h e r e b y facilitating isolation. A l t h o u g h all g r o w t h a p p e a r s b r o w n ish grey on chloral h y d r a t e , on c o m p l e t e m e d i u m sectors w i t h d i f f e r e n t colours are e x t r e m e l y p r o m i n e n t because of
Fig. 2. The diploid was first streaked on complete medium + 0.02 M chloral hydrate, incubated for 5 days and then transferred to complete medium. After 2-3 days of the transfer, fast growing and profusely conidiating sectors are visible.
profuse conidiation. Out of 36 isolates picked up from s t r e a k s first g r o w n on chloral h y d r a t e a n d t h e n t r a n s ferred to c o m p l e t e m e d i u m , 35 t u r n e d o u t to be haploids a n d only one was a diploid (Table), i n d i c a t i n g t h e r e b y t h a t , d u r i n g 5 d a y s on chloral h y d r a t e , t h e r e is a l m o s t c o m p l e t e h a p l o i d i z a t i o n , following w h i c h t h e h a p l o i d sect o r s grow o u t of t h e streak. Using chloral h y d r a t e as t h e h a p l o i d i z i n g agent, 2 n e w l y i s o l a t e d F P A - r e s i s t a n t m u t a n t s (riboA 1, biA I; [flaX70 a n d riboA 1, biA L"/paX71) were assigned to t h e linkage g r o u p VI, a n d t h e t h i r d n e w isolate (riboA 1, biA 1;/paX72) was assigned to t h e linkage g r o u p i. These a s s i g n m e n t s were c o n f i r m e d b y using F P A as t h e h a p l o i d i z i n g a g e n t 9. S e g r e g a t i o n of all o t h e r m a r k e r s was as e x p e c t e d on t h e basis of t h e i r linkage g r o u p locations. T h e diploid b e t w e e n / p a X 7 0 a n d M S E gave o u t o n l y g r e e n sectors w h e n F P A was used as t h e h a p l o i d i z i n g agent, b u t on choral h y d r a t e well growing a n d p r o f u s e l y c o n i d i a t i n g yellow, w h i t e a n d g r e e n sectors could easily be o b t a i n e d . The above-mentioned chemical and the procedure make t h e i n d u c t i o n a n d isolation of h a p l o i d s f r o m a diploid less t i m e c o n s u m i n g a n d v e r y easy, t h u s facilitating t h e entire p r o c e d u r e of genetic m a p p i n g b a s e d on m i t o t i c segregation. Also, chloral h y d r a t e is m u c h c h e a p e r t h a n p-fluorop h e n y l a l a n i n e . I t is u n i q u e because of its dual f u n c t i o n i n d u c t i o n of c-mitosis a n d p o l y p l o i d y in higher p l a n t s and h a p l o i d i z a t i o n in Aspergillus. To our knowledge, t h e r e is no r e p o r t of efficient i n d u c t i o n of h a p l o i d i z a t i o n b y a n y of t h e polyploidizing a g e n t s in a n y organism. Thus, chloral h y d r a t e d e s e r v e s m o r e a t t e n t i o n as a p o t e n t i a l h a p l o i d i z i n g a g e n t for p l a n t a n d a n i m a l ceils. Since it is a w i d e l y used drug, such i n v e s t i g a t i o n s would a d d to our k n o w l e d g e r e g a r d i n g its effects on h u m a n beings.
1 A. LEWAN,Hereditas 2d, 471 (1938). 2 H. Ris, Biol. Bull. 96, 90 (1949). 3 p. SENTEIN, Chromosoma dS, 215 (1974). 4 B. M~RCER and N. R. MORRIS, J. gen. Mierobiol. 88, 197 (1975). 5 j. A. ROPER, Experientia 8, 14 (1952). G. PONTECORVO,A. Rev. Microbiol. 70, 393 (1956). U. SINHA, Genetics 62, 495 (1969). 8 K. S. MCCULLYand E. FORBES, Genet. Res. 6, 352 (1965). 9 p. LHOAS, Nature, Lond. 790, 794 (1961).
