Plant Cell Reports
Plant Cell Reports (1986) 5:273-275
© Springer-Verlag 1986
Pollen embryogenesis in anther cultures of Solanum carolinense L. Thomas L. Reynolds Department of Biology, The University of North Carolina, Charlotte, NC 28223, USA Received February 25, 1986 - Communicated by F.Constabel
ABSTRACT
MATERIAL AND METHODS
The d i r e c t d i f f e r e n t i a t i o n of b i c e l l u l a r pollen grains of Solanum carolinense L. (Horse-nettle; Solanaceae) into embryoids and p l a n t l e t s was induced by c u l t u r i n g whole anthers on Murashige and Skoog's medium supplemented with IAA. The highest frequency o f embryogenic induction occurred at 10 mg/l IAA. Developmentally, both the generative and vegetative cells of the pollen grain contributed to embryoid formation whose pattern of development was s i m i l a r to that of zygotic embryos. In a previous study, i t was shown that 2,4-D promoted callus formation by pollen grains in cultured anthers of S. carolinense. I t appears then that there are two d i s t i n c t pathways of androgenesis in this species that are determined by the type of auxin present in the medium.
Plants o f Solanum carolinense used in this study were grown from seed in a greenhouse as described by Reynolds (1984). Flower buds containing b i c e l l u l a r pollen grains were surface s t e r i l i z e d in 12% (v/v) commercial bleach for 5 min and washed several times in s t e r i l e d i s t i l l e d water. Whole anthers from a single flower bud were cultured in 5 cm diameter petri dishes containing 10 ml of MS medium (Murashige and Skoog 1962) supplemented with 0.8% agar. Variations in the concentration of IAA added to the medium are given in the next section. Each culture contained four anthers; the f i f t h was squashed and stained with acetocarmine to determine the stage of pollen development. Inoculated plates were incubated in a growth chamber with an 18 hr photoperiod (120 #E m-2 s - l ) at 25 ~ 2 C. Morphogenic changes were followed by staining anther cultures at i n t e r v a l s with acetocarmine. For each experiment, at least 30 cultures were made in three replicates of I0 ( t o t a l of 120 anthers per treatment). To determine culture e f f i c i e n c y , 15-20 anthers were examined 12 days a f t e r culture and the number of m u l t i c e l l u l a r pollen grains and embryoids were counted using a compound microscope. This period was chosen as a compromise between the e l i m i n a t i o n of a small number of late d i v i d i n g pollen from counts and the d i f f i c u l t y o f making accurate counts at l a t e r periods due to the advanced stages o f development of embryoids in culture
Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, i n d o l e - 3 - a c e t i c acid; BA, benzyladenine; KIN, k i n e t i n ; MS, Murashige and Skoog. INTRODUCTION In a previous i n v e s t i g a t i o n (Reynolds 1984) i t was demonstrated that b i c e l l u l a r pollen grains of Solanum carolinense L. could be induced to form callus when anthers of this plant were cultured on medium containing 2,4-D. When subcultured onto medium lacking 2,4-D, the callus regenerated via organogenesis e x c l u s i v e l y . In a subsequent study concerning the morphogenic competence of somatic tissue cultures of Solanum carolinense, i t was shown that 2,4-D was also necessary for callus i n i t i a t i o n but had to be replaced by cytokinin before regeneration would occur (Reynolds 1986). In t h i s instance, the mode of regeneration was cytokinin dependent since high concentrations o f BA and a l l concentrations o f KIN promoted organogenesis while low concentrations of BA induced somatic embryogenesis in addition to organogenesis (Reynolds 1986). In the Solanaceae, the formation of pollen-derived embryos and p l a n t l e t s using anther culture techniques is a common feature (Raghavan 1976, Maheshwari et a l . 1982, Bajaj 1983). Since attempts to induce embryogenesis from p o l l e n - c a l l u s cultures of S. carolinense using 2,4-D and KIN were unsuccessful (Reynolds 1984), this i n v e s t i g a t i o n was i n i t i a t e d to examine the effects o f a second auxin, IAA, on pollen morphogenesis o f this species. The d i r e c t induction of pollen embryogenesis in anthers of S. carolinense is described in this paper.
RESULTS AND DISCUSSION As shown in the previous study (Reynolds 1984), b i c e l l u l a r pollen grains o f S. carolinense were most susceptible to androgenic induction. At this time, pollen grains were densely cytoplasmic and possessed d i s t i n c t generative and vegetative cell nuclei separated by a thin c e l l wall not v i s i b l e in whole mounts (Fig. 1). Acetocarmine squashes o f anthers cultured on MS medium containing IAA (1-15 mg/l) showed that within 72 hr, the majority o f the pollen grains did not take up s t a i n , These pollen often appeared empty and shrunken or enlarged and f i l l e d with starch and did not show any further development in culture. Other pollen at t h i s time were m u l t i c e l l u l a r and stained intensely with acetocarmine (Fig. 2). Both the generative and vegetative cells contributed to these p o t e n t i a l l y embryogenic c e l l masses which continued to divide within the anther to form typical globular and b i p o l a r embryoids (Figs. 3,4,5). Mature
274
,~
~FI~ ¸¸
Figs. 1-6. Pollen embryogenesis in Solanum carolinense, i . B i c e l l u l a r pollen with vegetative (v) and generative (g) nuclei at the time of anther culture. 2. M u l t i c e l l u l a r proembryoid 72 hrs a f t e r culture. 3. Young globular embryoids (arrows) I week a f t e r c u l t u r e . 4. Bipolar embryoid with organized radicle (r) 2 weeks a f t e r culture. 5. Torpedo-shaped embryoid with radicle (r) and cotyledons (c). 6. Developing shoot (Sh) of young plant emerging from anther (arrow) 3 weeks a f t e r culture. Calibration bars = I0 ~m.
embryoids were formed within 3 weeks a f t e r c u l t u r e i n o c u l a t i o n . With further development, embryoids broke through the anther wall and continued to d i f f e r e n t i a t e leaves and roots (Fig. 6). Plantlets were grown in culture u n t i l they could be transferred to vermiculite and soil mixture in the greenhouse. Chromosome counts of root t i p squashes of regenerated plants revealed t h e i r haploid nature (n = 12). The presence o f IAA was essential for embryogenesis. Increasing concentrations of IAA tended to enhance embryogenic induction with the highest frequency of embryoid formation observed at I0 mg/l (Table i ) . This i n v e s t i g a t i o n shows that haploid embryoids and p l a n t l e t s can be obtained from S. earolinense by c u l t u r i n g the anthers on medium containing IAA. In conjunction with previous observations, i t appears that there are two d i s t i n c t pathways of androgenesis in this species that are determined by the type of auxin present in the medium. As stated in the I n t r o duction, 2,4-D favors rapid c e l l p r o l i f e r a t i o n and callus formation while IAA stimulates d i r e c t embryoid production by pollen. The role of plant hormones in the induction of androgenesis is unclear. In Nicotiana (Nitsch 1969), Hyoscyamus (Raghavan 1975), and Datura (Sunderland et a l . 1974), pollen embryogenesis can be induced on a simple mineral-sucrose medium. However, Sopory and Maheshwari (1976) l a t e r showed that the addition of IAA to anther culture medium for Datura enhanced embryogenesis while 2,4-D had an i n h i b i t o r y e f f e c t . In a manner s i m i l a r to S. carolinense, pollen grains in anthers of Hyoscyamus niger cultured on 2,4-D containing medium w i l l form
Table 1. Embryoid induction frequency in anthers of
So~anum carolinense a f t e r 12 days in culture IAA concentration
Embryoid frequency b
(mg/l)a 0 1.0
11.2+4.5
3.0
29.9 + 8.0
5.0
51.1+6.2
7.0
44.8 + 9.3
10.0
77.3+8.9
15.0
63.0+7.7
a IAA added to MS medium supplemented with 0.8% agar b Embryoid frequency is the mean number of embryoids per anther from at least 40 anthers in two independent experiments ! standard error.
callus (Raghavan 1978). Unlike Solarium, pollenderived callus of H. niger regenerated Via organo-
275 genesis and embryogenesis when subcultured onto medium lacking 2,4-D (Raghavan and Nagmani 1983). The i n a b i l i t y to induce embryogenesis in pollen-derived callus cultures of S. c a r o l i ~ n s e may be due to differences in the developmental h i s t o r y of these c e l l s which could a l t e r t h e i r a b i l i t y to respond to a given hormonal signal since somatic callus cultures r e a d i l y form embryoids in addition to shoots and roots (Reynolds 1986). The o r i g i n and nature of these differences and t h e i r effects on morphogenesis in androgenic and somatic tissue cultures of Solo~um oarolinense are c u r r e n t l y under i n v e s t i g a t i o n .
ACKNOWLEDGEMENTS This study was supported in part by funds from the Foundation of the University of North Carolina at Charlotte and from the State of North Carolina.
REFERENCES Bajaj YPS (1983) In: Evans DA, Sharp WR, Ammirato PV, ¥amada Y (eds) Handbook of plant cell c u l t u r e , vol 1, Macmillan Publishing Co, New York, pp 228-287 Maheshwari SC, Rashid A, Tyagi AK (1982) Amer J Bot 69:865-879 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Nitsch JP (1969) Phytomorph 19: 389-40Raghavan V (1975) Z Pflanzenphysiol 76:89-92 Raghavan V (1976) Experimental embryogenesis in vascular plants, Academic Press, London Raghavan V (1978) Amer J Bot 65:984-1002 Raghavan V, Nagmani R (1983) Amer J Bot 70:524-531 Reynolds TL (1984) J Plant Physiol 117:157-161 Reynolds TL (1986) Amer J Bot 73:914-918 Sopory SK, ~aheshwari SC (1976) J Exp Bot 27:58-68 Sunderland N, Collins GB, Dunwell JM .(1974) Planta 117:227-241
Plant Cell Reports (1986) 5:273-275
© Springer-Verlag 1986
Pollen embryogenesis in anther cultures of Solanum carolinense L. Thomas L. Reynolds Department of Biology, The University of North Carolina, Charlotte, NC 28223, USA Received February 25, 1986 - Communicated by F.Constabel
ABSTRACT
MATERIAL AND METHODS
The d i r e c t d i f f e r e n t i a t i o n of b i c e l l u l a r pollen grains of Solanum carolinense L. (Horse-nettle; Solanaceae) into embryoids and p l a n t l e t s was induced by c u l t u r i n g whole anthers on Murashige and Skoog's medium supplemented with IAA. The highest frequency o f embryogenic induction occurred at 10 mg/l IAA. Developmentally, both the generative and vegetative cells of the pollen grain contributed to embryoid formation whose pattern of development was s i m i l a r to that of zygotic embryos. In a previous study, i t was shown that 2,4-D promoted callus formation by pollen grains in cultured anthers of S. carolinense. I t appears then that there are two d i s t i n c t pathways of androgenesis in this species that are determined by the type of auxin present in the medium.
Plants o f Solanum carolinense used in this study were grown from seed in a greenhouse as described by Reynolds (1984). Flower buds containing b i c e l l u l a r pollen grains were surface s t e r i l i z e d in 12% (v/v) commercial bleach for 5 min and washed several times in s t e r i l e d i s t i l l e d water. Whole anthers from a single flower bud were cultured in 5 cm diameter petri dishes containing 10 ml of MS medium (Murashige and Skoog 1962) supplemented with 0.8% agar. Variations in the concentration of IAA added to the medium are given in the next section. Each culture contained four anthers; the f i f t h was squashed and stained with acetocarmine to determine the stage of pollen development. Inoculated plates were incubated in a growth chamber with an 18 hr photoperiod (120 #E m-2 s - l ) at 25 ~ 2 C. Morphogenic changes were followed by staining anther cultures at i n t e r v a l s with acetocarmine. For each experiment, at least 30 cultures were made in three replicates of I0 ( t o t a l of 120 anthers per treatment). To determine culture e f f i c i e n c y , 15-20 anthers were examined 12 days a f t e r culture and the number of m u l t i c e l l u l a r pollen grains and embryoids were counted using a compound microscope. This period was chosen as a compromise between the e l i m i n a t i o n of a small number of late d i v i d i n g pollen from counts and the d i f f i c u l t y o f making accurate counts at l a t e r periods due to the advanced stages o f development of embryoids in culture
Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, i n d o l e - 3 - a c e t i c acid; BA, benzyladenine; KIN, k i n e t i n ; MS, Murashige and Skoog. INTRODUCTION In a previous i n v e s t i g a t i o n (Reynolds 1984) i t was demonstrated that b i c e l l u l a r pollen grains of Solanum carolinense L. could be induced to form callus when anthers of this plant were cultured on medium containing 2,4-D. When subcultured onto medium lacking 2,4-D, the callus regenerated via organogenesis e x c l u s i v e l y . In a subsequent study concerning the morphogenic competence of somatic tissue cultures of Solanum carolinense, i t was shown that 2,4-D was also necessary for callus i n i t i a t i o n but had to be replaced by cytokinin before regeneration would occur (Reynolds 1986). In t h i s instance, the mode of regeneration was cytokinin dependent since high concentrations o f BA and a l l concentrations o f KIN promoted organogenesis while low concentrations of BA induced somatic embryogenesis in addition to organogenesis (Reynolds 1986). In the Solanaceae, the formation of pollen-derived embryos and p l a n t l e t s using anther culture techniques is a common feature (Raghavan 1976, Maheshwari et a l . 1982, Bajaj 1983). Since attempts to induce embryogenesis from p o l l e n - c a l l u s cultures of S. carolinense using 2,4-D and KIN were unsuccessful (Reynolds 1984), this i n v e s t i g a t i o n was i n i t i a t e d to examine the effects o f a second auxin, IAA, on pollen morphogenesis o f this species. The d i r e c t induction of pollen embryogenesis in anthers of S. carolinense is described in this paper.
RESULTS AND DISCUSSION As shown in the previous study (Reynolds 1984), b i c e l l u l a r pollen grains o f S. carolinense were most susceptible to androgenic induction. At this time, pollen grains were densely cytoplasmic and possessed d i s t i n c t generative and vegetative cell nuclei separated by a thin c e l l wall not v i s i b l e in whole mounts (Fig. 1). Acetocarmine squashes o f anthers cultured on MS medium containing IAA (1-15 mg/l) showed that within 72 hr, the majority o f the pollen grains did not take up s t a i n , These pollen often appeared empty and shrunken or enlarged and f i l l e d with starch and did not show any further development in culture. Other pollen at t h i s time were m u l t i c e l l u l a r and stained intensely with acetocarmine (Fig. 2). Both the generative and vegetative cells contributed to these p o t e n t i a l l y embryogenic c e l l masses which continued to divide within the anther to form typical globular and b i p o l a r embryoids (Figs. 3,4,5). Mature
274
,~
~FI~ ¸¸
Figs. 1-6. Pollen embryogenesis in Solanum carolinense, i . B i c e l l u l a r pollen with vegetative (v) and generative (g) nuclei at the time of anther culture. 2. M u l t i c e l l u l a r proembryoid 72 hrs a f t e r culture. 3. Young globular embryoids (arrows) I week a f t e r c u l t u r e . 4. Bipolar embryoid with organized radicle (r) 2 weeks a f t e r culture. 5. Torpedo-shaped embryoid with radicle (r) and cotyledons (c). 6. Developing shoot (Sh) of young plant emerging from anther (arrow) 3 weeks a f t e r culture. Calibration bars = I0 ~m.
embryoids were formed within 3 weeks a f t e r c u l t u r e i n o c u l a t i o n . With further development, embryoids broke through the anther wall and continued to d i f f e r e n t i a t e leaves and roots (Fig. 6). Plantlets were grown in culture u n t i l they could be transferred to vermiculite and soil mixture in the greenhouse. Chromosome counts of root t i p squashes of regenerated plants revealed t h e i r haploid nature (n = 12). The presence o f IAA was essential for embryogenesis. Increasing concentrations of IAA tended to enhance embryogenic induction with the highest frequency of embryoid formation observed at I0 mg/l (Table i ) . This i n v e s t i g a t i o n shows that haploid embryoids and p l a n t l e t s can be obtained from S. earolinense by c u l t u r i n g the anthers on medium containing IAA. In conjunction with previous observations, i t appears that there are two d i s t i n c t pathways of androgenesis in this species that are determined by the type of auxin present in the medium. As stated in the I n t r o duction, 2,4-D favors rapid c e l l p r o l i f e r a t i o n and callus formation while IAA stimulates d i r e c t embryoid production by pollen. The role of plant hormones in the induction of androgenesis is unclear. In Nicotiana (Nitsch 1969), Hyoscyamus (Raghavan 1975), and Datura (Sunderland et a l . 1974), pollen embryogenesis can be induced on a simple mineral-sucrose medium. However, Sopory and Maheshwari (1976) l a t e r showed that the addition of IAA to anther culture medium for Datura enhanced embryogenesis while 2,4-D had an i n h i b i t o r y e f f e c t . In a manner s i m i l a r to S. carolinense, pollen grains in anthers of Hyoscyamus niger cultured on 2,4-D containing medium w i l l form
Table 1. Embryoid induction frequency in anthers of
So~anum carolinense a f t e r 12 days in culture IAA concentration
Embryoid frequency b
(mg/l)a 0 1.0
11.2+4.5
3.0
29.9 + 8.0
5.0
51.1+6.2
7.0
44.8 + 9.3
10.0
77.3+8.9
15.0
63.0+7.7
a IAA added to MS medium supplemented with 0.8% agar b Embryoid frequency is the mean number of embryoids per anther from at least 40 anthers in two independent experiments ! standard error.
callus (Raghavan 1978). Unlike Solarium, pollenderived callus of H. niger regenerated Via organo-
275 genesis and embryogenesis when subcultured onto medium lacking 2,4-D (Raghavan and Nagmani 1983). The i n a b i l i t y to induce embryogenesis in pollen-derived callus cultures of S. c a r o l i ~ n s e may be due to differences in the developmental h i s t o r y of these c e l l s which could a l t e r t h e i r a b i l i t y to respond to a given hormonal signal since somatic callus cultures r e a d i l y form embryoids in addition to shoots and roots (Reynolds 1986). The o r i g i n and nature of these differences and t h e i r effects on morphogenesis in androgenic and somatic tissue cultures of Solo~um oarolinense are c u r r e n t l y under i n v e s t i g a t i o n .
ACKNOWLEDGEMENTS This study was supported in part by funds from the Foundation of the University of North Carolina at Charlotte and from the State of North Carolina.
REFERENCES Bajaj YPS (1983) In: Evans DA, Sharp WR, Ammirato PV, ¥amada Y (eds) Handbook of plant cell c u l t u r e , vol 1, Macmillan Publishing Co, New York, pp 228-287 Maheshwari SC, Rashid A, Tyagi AK (1982) Amer J Bot 69:865-879 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Nitsch JP (1969) Phytomorph 19: 389-40Raghavan V (1975) Z Pflanzenphysiol 76:89-92 Raghavan V (1976) Experimental embryogenesis in vascular plants, Academic Press, London Raghavan V (1978) Amer J Bot 65:984-1002 Raghavan V, Nagmani R (1983) Amer J Bot 70:524-531 Reynolds TL (1984) J Plant Physiol 117:157-161 Reynolds TL (1986) Amer J Bot 73:914-918 Sopory SK, ~aheshwari SC (1976) J Exp Bot 27:58-68 Sunderland N, Collins GB, Dunwell JM .(1974) Planta 117:227-241
