THE INDUCTION OF ROOT AND SHOOT MORPHOGENESIS IN
Phaseolus vulgaris
TISSUE CULTURES*
O.J. CROCOMO** J.E:. P I T E R S * * * W . R . SHARP***
ABSTRACT
Exogenous concentrations of bean seed extract prepared from seeds pretreated in aerated water, homogenized in Veliky and Martin's 6 7 - V salt solution, filtered, and added to t h e culture medium at proper concentrations p r o m o t e callus proliferation, r o o t morphogenesis, and shoot morphogenesis in leaf explains of Phaseolus vulgaris var. Bico de O u r o . The activity of the bean seed factor is dependent upon the period of pretreatment in aerated water.
I N T R O D U C T I O N
If the science of plant tissue c u l t u r e is t o be applied t o t h e a d v a n c e m e n t of c r o p i m p r o v e m e n t , t h e i n d u c t i o n a n d c o n t r o l of m o r p h o g e n e s i s m u s t be o b t a i n e d . I l l L D E -B R A N D T et al. ( 1 9 6 3 ) a t t e m p t e d t o establish tissue c u l t u r e s of 3 2 species of agricul-tural p l a n t s , and a l t h o u g h success was o b t a i n e d in gaining callus from these species, only pea and endive resulted in differentiated plantlets. Tissue c u l t u r e s of Phaseolus vulgaris proved especially difficult with n o differentiation and n o c h l o r p p h y l pro-d u c t i o n . Since this w o r k , Phaseolus tissue c u l t u r e has been limited t o m e t a b o l i c studies ( L A M P O R T , 1 9 6 4 ; GAM Β O R G , 1 9 6 6 ; M E H T A et al., 1 9 6 7 ; LIAU & B O L L , 1 9 7 1 ; VLL1KY, 1 9 7 2 ) e x c e p t for a s t u d y of vascular differentiation c o n t r o l l e d by indole acetic acid (I A A) and sucrose ( J E F F S & N O R T H C O T E , 1 9 6 7 ) . At the Eucarpia Con-ference, Aseptic C u l t u r e M e t h o d s in Plant Breeding held at the University of Leeds. Leeds, England, J u l y 9 - 1 3 , 1 9 7 3 , it was recognized that t h e p o t e n t i a l application of cell c u l t u r e t e c h n i q u e t o e c o n o m i c plant i m p r o v e m e n t is limited in most tissue culture s y s t e m s because of o u r inability t o c o n t r o l m o r p h o g e n e s i s .
R e c e n t w o r k in Phaseolus using various a u x i n / c y t o k i n i n c o m b i n a t i o n s d e m o n s -t r a -t e d c o n -t r o l o f callus g r o w -t h a n d limi-ted c o n -t r o l of r o o -t m o r p h o g e n e s i s , bu-t no s h o o t f o r m a t i o n was i n d u c e d ( C R O C O M O , S H A R P & P E T E R S , 1976). Excellent c o n t r o l of r o o t m o r p h o g e n e s i s was o b t a i n e d with the a d d i t i o n of e x o g e n o u s nicotine to t h e n u t r i e n t w h e n k i n e t i n was r e m o v e d ( P E T E R S et al., 1 9 7 6 ) . Unsuccessful a t t e m p t s
* Received for publication in 1 0 / 1 0 / 1 9 7 5 , Supported by grants from Comissão Nacional de Energia Nuclear (CNEN) and Fundação de Amparo à Pesquisa do Listado de São Paulo (FAPESP)
-Brazil.
at o b t a i n i n g shoot m o r p h o g e n e s i s with a defined n u t i i e n t p r o m p t e d t h e r e t u r n t o undefined n u t r i e n t s u p p l e m e n t s . This r e p o r t p e r t a i n s t o t h e a d d i t i o n of a b e e n seed extract for i n d u c t i o n o f p l a n t l e t m o r p h o g e n e s i s . Similar e x p e r i m e n t a t i o n has been p e r f o r m e d using a plant e x t r a c t s u p p l e m e n t for t h e i n d u c t i o n o f e m b r y o g e n e i s in t o m a t o cell c u l t u r e s ( D E B E R G H & N I T S C H , 1 9 7 3 ) .
M A T E R I A L S A N D M E T H O D S
Leaves from 2-wk old Phaseolus vulgaris var. Bico de O u r o were surface sterilized in 20% v/v h y p o c h l o r i t e for 15 m i n . F o l l o w i n g t w o rinses in sterile distilled H20 , t h e
leaves were sectioned i n t o 5 m m squares and i n o c u l a t e d in 20 χ 150 m m test t u b e s c o n t a i n i n g t e n ml of n u t r i e n t . C u l t u r e s were g r o w n for 8 w e e k s u n d e r c o n s t a n t en-v i r o n m e n t a l c o n d i t i o n s ( 2 5 ° C ; 12-h p h o t o p e r i o d ; illumination from cool-white fluo-rescent lamps giving ca. 2 0 0 ft-c o u t s i d e t h e c u l t u r e b o t t l e s ) .
All n u t r i e n t c o n t a i n e d Veliky and Martin's ( 1 9 7 0 ) 6 7 - V salts and vitamins. Follow-ing a d d i t i o n of a u x i n , c y t o k i n i n a n d b e a n e x t r a c t t h e m e d i u m was adjusted t o a final pH o f 4 . 5 , solidified with 1% agar a n d sterilized for 15 min at 1 2 1 ° C .
T h e e x t r a c t s consisted of Phaseolus vulgaris var. Carioca bean seeds h o m o g e n i z e d in 6 7 - V salts at c o n c e n t r a t i o n s ranging from 1/100 bean seed/ml t o 1 bean s e e d / m l . Seeds were soaked in aerated t a p w a t e r prior to h o m o g e n i z a t i o n and t h e e x t r a c t was s u b s e q u e n t l y filtered t h r o u g h cheese c l o t h .
T h e following tests were designed with variations in t h e a u x i n / c y t o k i n i n con-c e n t r a t i o n , and in the con-c o n con-c e n t r a t i o n o f bean seed e x t r a con-c t of various ages.
1 - Test of effect of various kinetin c o n c e n t r a t i o n s in 6 7 - V m e d i u m plus bean e x t r a c t . In this test. IAA was c o n s t a n t at a c o n c e n t r a t i o n of 1.0 mg/1 and kinetin varied from 0 - 1 0 mg/1. T h e e x t r a c t a d d i t i o n consisted of a h o m o g e n a t e of bean seeds soaked for o n e d a y at a c o n s t a n t c o n c e n t r a t i o n of 1/4 b e a n / m l .
2 — Test of age of soaked bean and bean e x t r a c t c o n c e n t r a t i o n . Auxin and c y t o k i n i n c o n c e n t r a t i o n s were c o n s t a n t ( I A A = 2.0 mg/1, N A A = 1.0 mg/1 and kinetin = 0.2 mg/1). Bean e x t r a c t a d d i t i o n s consisted of c o n c e n t r a t i o n s ranging from 1/100 bean seed/ml t o 1 bean s e e d / m l . A second variable in bean e x t r a c t c o m p o s i t i o n was the age of t h e b e a n . Beans soaked for 0 ( B E C - 0 ) , 1 ( B E - l ) , 3 (BE-3) and 7 (BE-7) days were a d d e d at the various c o n c e n t r a t i o n s .
Scores for callus proliferation a n d r o o t p r o d u c t i o n were averaged and t h e s t a n d a r d e r r o r of t h e m e a n c a l c u l a t e d . In the case of r o o t m o r p h o g e n e s i s , t h e m e a n was used as a " r o o t m o r p h o g e n e s i s i n d e x " since it represents b o t h p e r c e n t a g e of r o o t i n d u c t i o n and a m o u n t o f g r o w t h .
R E S U L T S
When e x o g e n o u s auxin and c y t o k i n i n c o n c e n t r a t i o n s are k e p t c o n s t a n t t h e addition o f various aged bean seed e x t r a c t t o the m e d i u m has little effect on callus growth (Fig. 4) e x c e p t for a possible inhibition of g r o w t h with 1-day old seed extract and for the higher c o n c e n t r a t i o n s at 1/2. 3/4 and 1 bean seed/ml of seeds 1-day old and older. A strong inhibition of r o o t m o r p h o g e n e s i s is p r o d u c e d by c o n c e n t r a t i o n s greater than
1/4 bean seed per nil e x c e p t for 0-day old bean seeds ( F i g . 5 ) .
DISCUSSION
The induction of shoot morphogenesis and the enhancement of callus growth and
root induction by the addition of a bean seed extract are significant advancements in
the culture of Phaseolus vulgaris. In extensive tests using 67—V salts and the auxin,
indole acetic acid (IAA) or napthalene acetic acid (NAA) accompanied by the
cyto-kines kinetin or zeatin, no plantlet morphogenesis was induced in more than 3,000
cultures (CROCOMO et al., 1976). Both callus growth and root morphogenesis could
be controlled by auxin and cytokinin concentration, but the levels obtained with the
addition of bean seed extract are significantly increased.
The work testing the age of the bean seed extract although preliminary in nature,
is both exciting and promising. It is our belief that morphogenesis in Phaseolus vulgaris
is very desirable and should be sought even if the return to undefined nutrient is
necessary. The use of the bean extract has been the most promising area of our attempts,
although another potentially significant morphogen in our system has been the addition
of nicotine to induce and control root morphogenesis (PETERS et al., 1976).
The potential application of tissue culture in the production of improved Phaseolus
varieties has been limited by the lack of morphogenesis. Since the significance of this
plant as a source of protein is becoming increased with the increased world food
short-ages, the induction of morphogenesis is very important. Haploid callus has been
obtain-ed in Phaseolus vulgaris (SHARP et al., unpublishobtain-ed) and the control of morphogenesis
in this system supplemented by promising protoplast work offers unlimited
possibili-ties for the production of new variepossibili-ties.
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