Plant Cell Reports
Plant Cell Reports (1985) 4: 28 - 30
© Springer-Verlag 1985
Anther culture of papaya
(CaricapapayaL.)
H. S. Tsay and C.Y. Su Department of Agronomy, Taiwan Agricultural Research Institute, Taichung, Taiwan 431, ROC Received October 30, 1985 / Revised version received January 2, 1985 - Communicated by J. Widholm
ABSTRACT Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid Dlantlets and pollen-derived embryoids were Obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.
inositol and 6% sucrose. Growth regulators including NAA, BA, kinetin and 2,4-D were added to the basal medium in various concentrations and selected combi-
Abbreviations MS NAA BA 2,4-D
~urashige and Skoog (1962) naphthaleneacetic acid 6-benzyladenine 2,4-dichloro~henoxyacetic acid INTRODUCTION
Papaya is one of the most important tropical fruits in Taiwan. However, its yield and quality are adversely affected by the prevalent virus diseases. There are no effective preventive methods or treatments against these diseases nor are resistant varieties available. The present study on papaya anther culture was attempted to gain an understanding of the process and relevent factors for haploid callus and plant production for mutation breeding or somatic hybridization work. >IATERIALS AND METHODS Anthers of three papaya varieties, Caric_aa2a_p_9_X~ L. cv. Sunrise, Tainung-CY and Florida (ring spot virus tolerant) were used as plant materials. Unopened flower buds were removed from donor plants and were treated with 70% alcohol for 30 sec, 0.5% sodium hypochlorite for 5 min and then washed several times in autoclaved distilled water. Anthers in different developmental stages were aseptically removed from flower buds and inoculated into 150 x 25 mm test tubes each containing i0 ml of MS mineral salts and vitamins supplemented with 0.4 mg/l thiamine HCI, i00 mg/l
Offprint requests to: H. S. Tsay
Figure i. Haploid plant emerging fro~ a cultured an o~o ther of papaya Figure 2. Secondary embryoids (SE) formed from the outer cells of the anther-derived embryoids (H:heart shape embryoid) Figure 3. Pollen-derived embryoid (E) directly induced from a cultured anther of papaya Figure 4. Multiplying papaya pollen-derived on MS medium with 3% sucrose Figure 5. Haploid plants regenerated derived embryoids.
embryoids
from the pollen-
29 Table i. The percent callus formation after 120 days from anthers of three papaya varieties cultured at different microspore developmental stages Microspore developmental stage
No. of anthers Sunrise
Tetrad Uninucleate Mitosis Early-binucleate ~ t u r e pollen
cultured
Tainung-CY
500 2500 1500 ii00 500
300 700 1250 500 500
Basal medium: (
1/2 MS salts and vitamins 6% sucrose and 1% agar. ):percentage
No. of anthers producing callus
Florida
Sunrise
Tainung-CY
300 300 300 300 300
1(0.2) 24(0.96) 29(1.93) 5(0.45) 0
0 8(1.14) 15(1.2) 2(0.4) 0
Florida 0 0 5(1.67) 0 0
supplemented with 2 mg/l NAA, 1 mg/l BA,
Table 2. The effects of growth regulators and light versus dark on callus and embryoid formation from papaya anthers cultured at the uninucleate pollen stage (cv. Tainung-CY) No. of anthers
No. of anthers producing
callus
No. of anther producing embryoids
Dark
Light
Dark
2(0.67) 0 0 0 0 19(0.63)
0 0 0 0 0 0
Growth regulators cultured NAA
BA
2
1 1 2 1 0 0
0 4 0 0 1
2,4-D
kinetin
-
-
mg/l
0 0 0 1 0 0
0 0 0 0 0 4
Dark
Light
700 300 750 300 300 2960
300 300 750 300 300 3000
8(1.14) 1(0.33) 2(0.27) 1(0.33) 0 27(0.91)
Duration of anther culture:120 days Basal medium:MS salts and vitamins supplemented ( ):percentage
Light 0 0 0 0 2(0.67) 0
with 3% sucrose and 1% agar
nations. After adjusting the pH value to 5.7 + 0.i, 0.8% Phytagar (Gibco) was added. The medium was autoclaved at 1.2 kg/cm for 15 min. Cultures were maintained in a culture room at 27°+ ~ C and under 16 h daily illumination with 1,500 lux fluorescent light for embryogenesis or in the dark for callus formation. Anthers were squashed in a drop of 1% acetocarmine or pretreated by strong acid (Tsay 1980) to determine the microspore developmental stages. For histological studies anther-derived embryoids were fixed in FAA (7 ml of 40% formaldehyde : 3 g o f acetic acid : 90 ml of 70% ethanol), dehydrated through the TBA series and embedded in Tissue Prep paraffin. Sections were cut at I0 ym, stained in Heidenhains' iron hematoxylin (Reid et al., 1950) and examined under a light microscope. RESULTS Papaya anthers containing microspores at the tetrad to early binucleate stages were successfully cultured on 1/2 strength MS inorganic salts and vitamins supplemented with full strength Na-Fe-EDTA and organic substances, 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus formation (Table I). The highest frequency of callus induction was obtained from anthers with uninucleate to first pollen mitosis microspores (Table i) from all three varieties. Antherderived callus was induced in both dark and light conditions although better results were obtained when anther were cultured On MS medium with 2 mg/l NAA and 1 mg/l BA or 1 mg/l NAA and 4 mg/l kinetin in darkness (Table 2). ~ e n calli were transferred to different media combinations, some pigmentation changes including green spots occurred, but no organ differentiation was noted. However, haploid plants (Figure I) or embryoids (Figure 3) were induced directly from anthers cultured on MS basal medium with 3% sucrose
without growth regulators under low light intensity
(1,5oo). More embryoids formed on the surface when antherderived embryoids were transferred to MS medium with 3% sucrose without growth regulators. Histological studies showed that secondary embryoids were continuously produced by the outer cells on the original anther-derived embryoids (Figure 2). These secondary embryoids could develop into either embryoids (Figure 4) or haploid plants (Figure 5). Approximately 50 haploid p!antlets have been induced from these embryoids. Cytological examination of root tip cells of about 20 embryoid-derived plantlets showed a chromosome number of n=x=9 in all cases. DISCUSSION Anther-derived haploid plants can be induced via direct embryogenesis or indirect callus regeneration (Sunderland and Wick 1969, 1971). Guha and Maheshwari (1966, 1967) reported that Datura anthers cultured on growth regulator-containing medium always induced callus while embryoids could be produced using medium without growth regulators. Our results showed that papaya anther cultures behave similarly. Growth regulators not only induce microspore-derived callus, but also induce somatic cell-derived callus from tobacco connective tissue (Sunderland and Wick 1969, 1971). Our studies indicate that growth regulators should not be used if embryogenesis is desired from papaya anther cultures. Litz and Conover (1978) have reported that microspore-derived haploid plantlets could be induced from papaya anthers taken from 8-16 mm long inflorecence buds. The anthers were cultured in stationary liquid ~IS medium containing 3% surcose, 1% activated charcoal, 2.0 ~ m BA and 0.5 jam NAA. This procedure has been attempted many times in our laboratory, but
30 all anthers turned white and died. The reason for our inability to duplicate this work is not known. The method described in this paper can both induce microspore-derived embryoids from cultured anthers and also propagate these embryoids rapidly. If single cells or protoplasts can be isolated from these haploid emhryoids and embryoid-derived callus. These protoplasts can then be used for fusion or mutation breeding. Effort are now being concentrated on establishing haploid cultures from anthers or ring spot virus resistant Carica stipulata which is sexually imcompatible with C. papaya.
REFERENCES Guha S, Maheshwari
SC (1966) Nature 5057:97-98.
Guha S, Maheshwari SC (1967) Panchanan Maheshwari ~½morial:454-461. Litz RE, Conover RA (1978) Proc. Fla. State Hort. Soc. 91:180-192. Murashige 497.
T, Skoog F (1962) Physiol.
Plant.
15:473-
Reid ~ , Muriel VB, Thelda IA (1950) Plant microtechnique manual. Univ. of Calif. Davis, Calif. Sunderland N, Wicks FM (1969) Nature 224:1227-1229. Sunderland N, Wicks FM (1971) J. Exp. Bot. 22:216226. Tsay HS (1980) J. Agric. Res. China 29(2):103-106.
Plant Cell Reports (1985) 4: 28 - 30
© Springer-Verlag 1985
Anther culture of papaya
(CaricapapayaL.)
H. S. Tsay and C.Y. Su Department of Agronomy, Taiwan Agricultural Research Institute, Taichung, Taiwan 431, ROC Received October 30, 1985 / Revised version received January 2, 1985 - Communicated by J. Widholm
ABSTRACT Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid Dlantlets and pollen-derived embryoids were Obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.
inositol and 6% sucrose. Growth regulators including NAA, BA, kinetin and 2,4-D were added to the basal medium in various concentrations and selected combi-
Abbreviations MS NAA BA 2,4-D
~urashige and Skoog (1962) naphthaleneacetic acid 6-benzyladenine 2,4-dichloro~henoxyacetic acid INTRODUCTION
Papaya is one of the most important tropical fruits in Taiwan. However, its yield and quality are adversely affected by the prevalent virus diseases. There are no effective preventive methods or treatments against these diseases nor are resistant varieties available. The present study on papaya anther culture was attempted to gain an understanding of the process and relevent factors for haploid callus and plant production for mutation breeding or somatic hybridization work. >IATERIALS AND METHODS Anthers of three papaya varieties, Caric_aa2a_p_9_X~ L. cv. Sunrise, Tainung-CY and Florida (ring spot virus tolerant) were used as plant materials. Unopened flower buds were removed from donor plants and were treated with 70% alcohol for 30 sec, 0.5% sodium hypochlorite for 5 min and then washed several times in autoclaved distilled water. Anthers in different developmental stages were aseptically removed from flower buds and inoculated into 150 x 25 mm test tubes each containing i0 ml of MS mineral salts and vitamins supplemented with 0.4 mg/l thiamine HCI, i00 mg/l
Offprint requests to: H. S. Tsay
Figure i. Haploid plant emerging fro~ a cultured an o~o ther of papaya Figure 2. Secondary embryoids (SE) formed from the outer cells of the anther-derived embryoids (H:heart shape embryoid) Figure 3. Pollen-derived embryoid (E) directly induced from a cultured anther of papaya Figure 4. Multiplying papaya pollen-derived on MS medium with 3% sucrose Figure 5. Haploid plants regenerated derived embryoids.
embryoids
from the pollen-
29 Table i. The percent callus formation after 120 days from anthers of three papaya varieties cultured at different microspore developmental stages Microspore developmental stage
No. of anthers Sunrise
Tetrad Uninucleate Mitosis Early-binucleate ~ t u r e pollen
cultured
Tainung-CY
500 2500 1500 ii00 500
300 700 1250 500 500
Basal medium: (
1/2 MS salts and vitamins 6% sucrose and 1% agar. ):percentage
No. of anthers producing callus
Florida
Sunrise
Tainung-CY
300 300 300 300 300
1(0.2) 24(0.96) 29(1.93) 5(0.45) 0
0 8(1.14) 15(1.2) 2(0.4) 0
Florida 0 0 5(1.67) 0 0
supplemented with 2 mg/l NAA, 1 mg/l BA,
Table 2. The effects of growth regulators and light versus dark on callus and embryoid formation from papaya anthers cultured at the uninucleate pollen stage (cv. Tainung-CY) No. of anthers
No. of anthers producing
callus
No. of anther producing embryoids
Dark
Light
Dark
2(0.67) 0 0 0 0 19(0.63)
0 0 0 0 0 0
Growth regulators cultured NAA
BA
2
1 1 2 1 0 0
0 4 0 0 1
2,4-D
kinetin
-
-
mg/l
0 0 0 1 0 0
0 0 0 0 0 4
Dark
Light
700 300 750 300 300 2960
300 300 750 300 300 3000
8(1.14) 1(0.33) 2(0.27) 1(0.33) 0 27(0.91)
Duration of anther culture:120 days Basal medium:MS salts and vitamins supplemented ( ):percentage
Light 0 0 0 0 2(0.67) 0
with 3% sucrose and 1% agar
nations. After adjusting the pH value to 5.7 + 0.i, 0.8% Phytagar (Gibco) was added. The medium was autoclaved at 1.2 kg/cm for 15 min. Cultures were maintained in a culture room at 27°+ ~ C and under 16 h daily illumination with 1,500 lux fluorescent light for embryogenesis or in the dark for callus formation. Anthers were squashed in a drop of 1% acetocarmine or pretreated by strong acid (Tsay 1980) to determine the microspore developmental stages. For histological studies anther-derived embryoids were fixed in FAA (7 ml of 40% formaldehyde : 3 g o f acetic acid : 90 ml of 70% ethanol), dehydrated through the TBA series and embedded in Tissue Prep paraffin. Sections were cut at I0 ym, stained in Heidenhains' iron hematoxylin (Reid et al., 1950) and examined under a light microscope. RESULTS Papaya anthers containing microspores at the tetrad to early binucleate stages were successfully cultured on 1/2 strength MS inorganic salts and vitamins supplemented with full strength Na-Fe-EDTA and organic substances, 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus formation (Table I). The highest frequency of callus induction was obtained from anthers with uninucleate to first pollen mitosis microspores (Table i) from all three varieties. Antherderived callus was induced in both dark and light conditions although better results were obtained when anther were cultured On MS medium with 2 mg/l NAA and 1 mg/l BA or 1 mg/l NAA and 4 mg/l kinetin in darkness (Table 2). ~ e n calli were transferred to different media combinations, some pigmentation changes including green spots occurred, but no organ differentiation was noted. However, haploid plants (Figure I) or embryoids (Figure 3) were induced directly from anthers cultured on MS basal medium with 3% sucrose
without growth regulators under low light intensity
(1,5oo). More embryoids formed on the surface when antherderived embryoids were transferred to MS medium with 3% sucrose without growth regulators. Histological studies showed that secondary embryoids were continuously produced by the outer cells on the original anther-derived embryoids (Figure 2). These secondary embryoids could develop into either embryoids (Figure 4) or haploid plants (Figure 5). Approximately 50 haploid p!antlets have been induced from these embryoids. Cytological examination of root tip cells of about 20 embryoid-derived plantlets showed a chromosome number of n=x=9 in all cases. DISCUSSION Anther-derived haploid plants can be induced via direct embryogenesis or indirect callus regeneration (Sunderland and Wick 1969, 1971). Guha and Maheshwari (1966, 1967) reported that Datura anthers cultured on growth regulator-containing medium always induced callus while embryoids could be produced using medium without growth regulators. Our results showed that papaya anther cultures behave similarly. Growth regulators not only induce microspore-derived callus, but also induce somatic cell-derived callus from tobacco connective tissue (Sunderland and Wick 1969, 1971). Our studies indicate that growth regulators should not be used if embryogenesis is desired from papaya anther cultures. Litz and Conover (1978) have reported that microspore-derived haploid plantlets could be induced from papaya anthers taken from 8-16 mm long inflorecence buds. The anthers were cultured in stationary liquid ~IS medium containing 3% surcose, 1% activated charcoal, 2.0 ~ m BA and 0.5 jam NAA. This procedure has been attempted many times in our laboratory, but
30 all anthers turned white and died. The reason for our inability to duplicate this work is not known. The method described in this paper can both induce microspore-derived embryoids from cultured anthers and also propagate these embryoids rapidly. If single cells or protoplasts can be isolated from these haploid emhryoids and embryoid-derived callus. These protoplasts can then be used for fusion or mutation breeding. Effort are now being concentrated on establishing haploid cultures from anthers or ring spot virus resistant Carica stipulata which is sexually imcompatible with C. papaya.
REFERENCES Guha S, Maheshwari
SC (1966) Nature 5057:97-98.
Guha S, Maheshwari SC (1967) Panchanan Maheshwari ~½morial:454-461. Litz RE, Conover RA (1978) Proc. Fla. State Hort. Soc. 91:180-192. Murashige 497.
T, Skoog F (1962) Physiol.
Plant.
15:473-
Reid ~ , Muriel VB, Thelda IA (1950) Plant microtechnique manual. Univ. of Calif. Davis, Calif. Sunderland N, Wicks FM (1969) Nature 224:1227-1229. Sunderland N, Wicks FM (1971) J. Exp. Bot. 22:216226. Tsay HS (1980) J. Agric. Res. China 29(2):103-106.
