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Rapid in vitro propagation of cauliflower
Plant Science, 90 (1993) 175-178 Elsevier Scientific Publishers Ireland Ltd.
175
Rapid in vitro propagation of cauliflower Atul Kumar, Vandana A. Kumar and Jitendra Kumar G.B. Pant University of Agriculture and Technology, Hill Campus. Ranichauri, Dt. Tehri Garhwal (India) (Received September 1lth, 1992; revision received January 19th, 1993; accepted January 22nd 1993)
A rapid single step method for complete plantlet regeneration in cauliflower is described. Curd explants cultured on Murashige and Skoog (MS) medium supplemented with 1 mg I-I indoleacetic acid (IAA) developed complete plantlets in 25 days. Regeneration potential of curd explants was drastically reduced with increasing storage of cauliflower curds at room temperature beyond 8 days of harvesting. Light intensity of 3000 lux supplied by white fluorescent tubes was optimum to evoke the best response. It was possible to field transfer the hardened plantlets within 35 days of culture initiation.
Key words: clonal; cauliflower curd; protocol
Introduction
Clonal propagation of cauliflower (Brassica oleracea var. botrytis L.) has been successfully attempted using mature curd tissue as the explant and various protocols [I-3] have been recommended. Using these protocols, it is possible to have clonal cauliflower plants in the field within 4 months of culture initation [11. This is a considerably long period, as following conventional nursery practice plants are ready for field transfer within 4 - 5 weeks of seed germination. Efforts at rapid in vitro regeneration in liquid medium [4] have resulted in vitrification of foliage [5] leading to problems in field transfer. Moreover, plantlets developing on liquid medium have to be transferred to artificial supports or agar-based medium to reduce vitrification [6] making the process tedious. We describe here a protocol through which complete plantlets can be regenerated in one step in only 25 days without vitrification of foliage and hardened plantlets are ready for field transfer Correspondence to: Atul Kumar, G.B. Pant University of Agri. and Tech., Hill Campus, P.O. Ranichauri, Distt. Tehri Garhwal, U.P., India.
within 35 days of culture initiation. Effects of hormonal composition, light intensity and explant age that influence rapidity are also investigated. Materials and Methods
Plant material Florets of approx. 1 cm diameter were excised from superficial inflorescence of cauliflower curd and surface-sterilized with 70% (v/v) ethanol for 90 s followed by commercial bleach solution (5%, w/w) containing 0.1% Tween-20 for 15 min. After three rinses with sterile distilled water, small (approx. 3 mm cubes) pieces of curd surface were dissected away from the explants and used to initiate cultures on semi-solid medium. Culture medium The culture medium consisted of Murashige and Skoog [7] macro and micro nutrients, vitamins, inositol, sucrose (3%, w/v) and phytagar (0.3%, w/v) as solidifying agent. All media used in these experiments were sterilized by autoclaving at 1 kg/cm and 120°C for 20 min. Culture flasks containing 30 ml of sterile medium were used for implantation of explants.
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd, Printed and Published in Ireland
176
Hormonal composition To optimize the hormonal composition for complete plantlet regeneration in one step, different combinations of indoleacetic acid (IAA) (1 mg 1-l alone and 0.5, 1.0 and 2.0 mg 1-1) and benzylaminopurine (BAP) (1 mg 1-I alone and 0.5 mg 1-1) were tried in MS medium.
Explant age Effect of explant age on regeneration potential was investigated by implanting curd pieces after 4, 8, 12 and 16 days of harvesting of cauliflower curd. After harvesting curds were kept at room temperature (20 4- 2°C) till the implantation was done.
Table I. Root and shoot development in cauliflower cultures under optimal conditions. Cultures were incubated under a 16h/day photoperiod at 28 ± 2°C (days) and 24 ± 2°C (nights). Response (after 25 days) Days taken for root initiation Rooting (%) No. roots/explant Length of roots (cm) No. shoots/explant Length of shoots (cm) No. leaves/explant
MS medium supplemented with IAA (1 mg l-I) a 7.4 ± 0.8 100 19.6 10.8 1.4 5.4 10.6
± 4± ± ±
2.1 1.4 0.3 0.6 1.5
aMean ± S.E. of three replications with 40 samples in the treatment.
Light &tensity Response of explants for shoot proliferation under different light intensities was studied at 1000, 2000, 3000 and 4000 lux under a 16-h/day photoperiod from white fluorescent tubes. Studies on the effects of explant age and light intensity were conducted by incubating cultures on MS medium supplemented with 1 mg 1-l IAA, the optimum concentration determined for plantlet regeneration.
Culture conditions Except for light intensity experiments, all cultures were incubated under a 16-h/day photoperiod and 8-h dark period at 28 ± 2°C during the day and 24 4- 2oc during the night with a light intensity of 3000 lux from white fluorescent tubes unless otherwise stated.
Hardening and field transfer The plantlets were transferred to a sterile soil and leaf mould mixture (3:1, w/w) in polythene bags kept covered with polythene sheet. These were grown in diffuse light with 16-h/day photoperiod for 5 days at 24 4- 2°C during the day and 20 ± 2°C during the night. Irrigation was done with Hoagland nutrient solution [8] as required. After 5 days the polythene cover was removed and plants were transferred to full light intensity in the laboratory and kept there for another 5 days. The experiments were repeated twice in three replications with 40 samples in each treatment and the data were subjected to statistical analysis.
Results and Discussion
Complete plantlets were regenerated from curd explants in 25 days in one step by supplementing MS medium with 1 mg 1-I IAA and incubating cultures under a 16-h/day photoperiod (3000 lux light intensity) at 28 ± 2°C (days) and 24 ± 2°C (nights) (Table I). After gradual hardening, hardened plants were field transferred with 80% survival rate within 35 days of culture initiation. Combinations of IAA with BAP and BAP alone induced rosetting of leaves and stunted growth of shoots (data not presented). In the protocols developed for plantlet regeneration using floral meristem as explant, different concentrations of plant hormones have been recommended in Linsmair and Skoog [9] medium i.e. 2.5 mg 1-I BA + 8 mg 1-1 IAA [4,10], 0.9/zM 2,4-D + 14/~M Kinetin or 0.44 I~M BA + 5.4/~M NAA [11,12]; or MS medium, i.e. 8 mg 1-1 IAA + 2.5 mg 1-I Kinetin [3]. It takes about 4 months to obtain a field transferred cauliflower plant population [1] and attempts to hasten shoot proliferation in liquid medium [4] have led to foliage vitrification [5] which poses problems during field transfer. In the present investigation, it was possible to regenerate complete plantlets in 25 days in one step and hardened plantlets were field transferred within 35 days of culture initiation. As cultures were grown on semi-solid (0.3% phytagar, w/v) medium, the growth was faster with no
177
lOC
z o nLI.I Z b_t W OE I'ILl d t-'-
z <
50
25
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13.
4 8 12 E X P L A NT A G E ('DAYS)
16
Fig. 1. Influence of explant age on plantlet regeneration potential after 25 days of incubation on MS medium supplemented with 1 mg 1-1 IAA. Mean 4- S.E. of three replications with 40 samples in each treatment.
Z O
100
12"~ ec hi LL .._1
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foliage vitrification. Developed plantlets were normal and healthy in appearence. Explants taken 4 and 8 days after harvesting o f cauliflower curd, stored at r o o m temperature after harvest, showed 100% regeneration potential while after 12 and 16 days o f harvesting, the response was reduced to 48% and 11%, respectively (Fig. 1). Thus regeneration potential o f curd explants decreased greatly with increasing storage o f cauliflower curds at r o o m temperature beyond 8 days o f harvesting. Influence o f light intensity on days taken for shoot proliferation and response of explants is shown in Fig. 2. Cultures incubated at a light intensity o f 4000 and 3000 lux t o o k only 3 days to initiate shoot proliferation with 100% o f explants responding. Cultures incubated at 2000 and 1000 lux took 8 and 12 days for initiating shoot proliferation and percentage o f responding explants was 64 and 42, respectively. The requirement for photoperiod and light intensity/quality for growing cultures has not been determined yet in cauliflower [1]. The present investigation suggests that 3000 lux light intensity supplied by white fluorescent tubes is o p t i m u m to evoke the best response. Following this protocol, it is possible to reduce the in vitro clonal propagation time from 4 months to approx. 35 days making it comparable to the time taken in conventional nursery practice. Plantlet regeneration at low I A A concentration in one step and without vitrification o f foliage will also make it economically feasible.
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Acknowledgements Authors are grateful to Dr. P.L. G a u t a m (Joint Director), Hill Campus, Ranichauri for guidance in the present investigation. References
o 1 LIGHT
2 3 4 INTENSITY (K LUX)
Fig. 2. Influenceof light intensity (K lux) on days taken for shoot proliferation (El) and % responding explants (121)after 25 days of incubation on MS medium supplemented with 1 mg 1-I IAA. Mean + S.E. of three replications with 40 samples in each treatment.
1 B.W.M. Grout, Cauliflower (Brassica oleracea var. botrytis L.), in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry Crops II, Vol. 6, Springer Verlag, 1988, pp. 211-225. 2 S.Y. Ze and B.B. Johnson, Cole Crops, in: P.V. Ammirato, D.A. Evans, W.R. Sharp and Y. Yamada (Eds.), Handbook of Plant Cell Culture Crop Species, Vol. 3, Macmillan, lnc, New York, London, 1984, pp. 227-246.
178 3
4
5
6
7
8
V.A. Kumar, A. Kumar and J. Kumar, In vitro plant regeneration of cauliflower (Brassica oleracea convar botrytis var botrytis) from mature curd. Indian J. Agric. Sci., 62 (1992) 429-431. D.G.A. Walkey and J.G.M. Woolfit, Rapid clonal multiplication of cauliflower by shake culture. J. Hort. Sci., 45 (1970) 205-206. B.W.M. Grout and P. Crisp, Practical aspects of the propagation of cauliflower by meristem culture. Acta Hort., 78 (1977) 289-296. K.C. Short, J. Warburton and A.V. Roberts, In vitro hardening of cultured cauliflower and chrysanthemum plantlets to humidity. Acta Hort., 212 (1987) 329-334. T. Murashige and F A. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15 (1962) 473-497. D.R. Hoagland, Lectures on the Inorganic Nutrition of
Plants, Chronica Botanica Company, Waltham, Mass. E.U. Linsmair and F.A. Skoog, Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18 (1965) 100-127. 10 P. Crisp and D.G.A. Walkey, The use of aseptic meristem culture in cauliflower breeding. Euphytica, 23 (1974) 305-313. 11 M. Buiatti, S. Baroncelli, A. Bennici, M. Pagliai and R. Tesi, Genetics of growth and differentiation in vitro of Brassica oleracea var. botrytis. II. An in vitro and in vivo analysis of a diallel cross. Z. Pflanzenphysiol., 72 (1974) 269-274. 12 M. Buiatti, S. Baroneelli and A. Bennici, Genetics of growth and differentiation in vitro of Brassica oleracea var. botrytis. IV. Genotype-hormone interactions. Z. Pflanzenphysiol., 73 (1974) 298-302. 9
175
Rapid in vitro propagation of cauliflower Atul Kumar, Vandana A. Kumar and Jitendra Kumar G.B. Pant University of Agriculture and Technology, Hill Campus. Ranichauri, Dt. Tehri Garhwal (India) (Received September 1lth, 1992; revision received January 19th, 1993; accepted January 22nd 1993)
A rapid single step method for complete plantlet regeneration in cauliflower is described. Curd explants cultured on Murashige and Skoog (MS) medium supplemented with 1 mg I-I indoleacetic acid (IAA) developed complete plantlets in 25 days. Regeneration potential of curd explants was drastically reduced with increasing storage of cauliflower curds at room temperature beyond 8 days of harvesting. Light intensity of 3000 lux supplied by white fluorescent tubes was optimum to evoke the best response. It was possible to field transfer the hardened plantlets within 35 days of culture initiation.
Key words: clonal; cauliflower curd; protocol
Introduction
Clonal propagation of cauliflower (Brassica oleracea var. botrytis L.) has been successfully attempted using mature curd tissue as the explant and various protocols [I-3] have been recommended. Using these protocols, it is possible to have clonal cauliflower plants in the field within 4 months of culture initation [11. This is a considerably long period, as following conventional nursery practice plants are ready for field transfer within 4 - 5 weeks of seed germination. Efforts at rapid in vitro regeneration in liquid medium [4] have resulted in vitrification of foliage [5] leading to problems in field transfer. Moreover, plantlets developing on liquid medium have to be transferred to artificial supports or agar-based medium to reduce vitrification [6] making the process tedious. We describe here a protocol through which complete plantlets can be regenerated in one step in only 25 days without vitrification of foliage and hardened plantlets are ready for field transfer Correspondence to: Atul Kumar, G.B. Pant University of Agri. and Tech., Hill Campus, P.O. Ranichauri, Distt. Tehri Garhwal, U.P., India.
within 35 days of culture initiation. Effects of hormonal composition, light intensity and explant age that influence rapidity are also investigated. Materials and Methods
Plant material Florets of approx. 1 cm diameter were excised from superficial inflorescence of cauliflower curd and surface-sterilized with 70% (v/v) ethanol for 90 s followed by commercial bleach solution (5%, w/w) containing 0.1% Tween-20 for 15 min. After three rinses with sterile distilled water, small (approx. 3 mm cubes) pieces of curd surface were dissected away from the explants and used to initiate cultures on semi-solid medium. Culture medium The culture medium consisted of Murashige and Skoog [7] macro and micro nutrients, vitamins, inositol, sucrose (3%, w/v) and phytagar (0.3%, w/v) as solidifying agent. All media used in these experiments were sterilized by autoclaving at 1 kg/cm and 120°C for 20 min. Culture flasks containing 30 ml of sterile medium were used for implantation of explants.
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd, Printed and Published in Ireland
176
Hormonal composition To optimize the hormonal composition for complete plantlet regeneration in one step, different combinations of indoleacetic acid (IAA) (1 mg 1-l alone and 0.5, 1.0 and 2.0 mg 1-1) and benzylaminopurine (BAP) (1 mg 1-I alone and 0.5 mg 1-1) were tried in MS medium.
Explant age Effect of explant age on regeneration potential was investigated by implanting curd pieces after 4, 8, 12 and 16 days of harvesting of cauliflower curd. After harvesting curds were kept at room temperature (20 4- 2°C) till the implantation was done.
Table I. Root and shoot development in cauliflower cultures under optimal conditions. Cultures were incubated under a 16h/day photoperiod at 28 ± 2°C (days) and 24 ± 2°C (nights). Response (after 25 days) Days taken for root initiation Rooting (%) No. roots/explant Length of roots (cm) No. shoots/explant Length of shoots (cm) No. leaves/explant
MS medium supplemented with IAA (1 mg l-I) a 7.4 ± 0.8 100 19.6 10.8 1.4 5.4 10.6
± 4± ± ±
2.1 1.4 0.3 0.6 1.5
aMean ± S.E. of three replications with 40 samples in the treatment.
Light &tensity Response of explants for shoot proliferation under different light intensities was studied at 1000, 2000, 3000 and 4000 lux under a 16-h/day photoperiod from white fluorescent tubes. Studies on the effects of explant age and light intensity were conducted by incubating cultures on MS medium supplemented with 1 mg 1-l IAA, the optimum concentration determined for plantlet regeneration.
Culture conditions Except for light intensity experiments, all cultures were incubated under a 16-h/day photoperiod and 8-h dark period at 28 ± 2°C during the day and 24 4- 2oc during the night with a light intensity of 3000 lux from white fluorescent tubes unless otherwise stated.
Hardening and field transfer The plantlets were transferred to a sterile soil and leaf mould mixture (3:1, w/w) in polythene bags kept covered with polythene sheet. These were grown in diffuse light with 16-h/day photoperiod for 5 days at 24 4- 2°C during the day and 20 ± 2°C during the night. Irrigation was done with Hoagland nutrient solution [8] as required. After 5 days the polythene cover was removed and plants were transferred to full light intensity in the laboratory and kept there for another 5 days. The experiments were repeated twice in three replications with 40 samples in each treatment and the data were subjected to statistical analysis.
Results and Discussion
Complete plantlets were regenerated from curd explants in 25 days in one step by supplementing MS medium with 1 mg 1-I IAA and incubating cultures under a 16-h/day photoperiod (3000 lux light intensity) at 28 ± 2°C (days) and 24 ± 2°C (nights) (Table I). After gradual hardening, hardened plants were field transferred with 80% survival rate within 35 days of culture initiation. Combinations of IAA with BAP and BAP alone induced rosetting of leaves and stunted growth of shoots (data not presented). In the protocols developed for plantlet regeneration using floral meristem as explant, different concentrations of plant hormones have been recommended in Linsmair and Skoog [9] medium i.e. 2.5 mg 1-I BA + 8 mg 1-1 IAA [4,10], 0.9/zM 2,4-D + 14/~M Kinetin or 0.44 I~M BA + 5.4/~M NAA [11,12]; or MS medium, i.e. 8 mg 1-1 IAA + 2.5 mg 1-I Kinetin [3]. It takes about 4 months to obtain a field transferred cauliflower plant population [1] and attempts to hasten shoot proliferation in liquid medium [4] have led to foliage vitrification [5] which poses problems during field transfer. In the present investigation, it was possible to regenerate complete plantlets in 25 days in one step and hardened plantlets were field transferred within 35 days of culture initiation. As cultures were grown on semi-solid (0.3% phytagar, w/v) medium, the growth was faster with no
177
lOC
z o nLI.I Z b_t W OE I'ILl d t-'-
z <
50
25
J
13.
4 8 12 E X P L A NT A G E ('DAYS)
16
Fig. 1. Influence of explant age on plantlet regeneration potential after 25 days of incubation on MS medium supplemented with 1 mg 1-1 IAA. Mean 4- S.E. of three replications with 40 samples in each treatment.
Z O
100
12"~ ec hi LL .._1
o 9 [3_
75 Of) l-
foliage vitrification. Developed plantlets were normal and healthy in appearence. Explants taken 4 and 8 days after harvesting o f cauliflower curd, stored at r o o m temperature after harvest, showed 100% regeneration potential while after 12 and 16 days o f harvesting, the response was reduced to 48% and 11%, respectively (Fig. 1). Thus regeneration potential o f curd explants decreased greatly with increasing storage o f cauliflower curds at r o o m temperature beyond 8 days o f harvesting. Influence o f light intensity on days taken for shoot proliferation and response of explants is shown in Fig. 2. Cultures incubated at a light intensity o f 4000 and 3000 lux t o o k only 3 days to initiate shoot proliferation with 100% o f explants responding. Cultures incubated at 2000 and 1000 lux took 8 and 12 days for initiating shoot proliferation and percentage o f responding explants was 64 and 42, respectively. The requirement for photoperiod and light intensity/quality for growing cultures has not been determined yet in cauliflower [1]. The present investigation suggests that 3000 lux light intensity supplied by white fluorescent tubes is o p t i m u m to evoke the best response. Following this protocol, it is possible to reduce the in vitro clonal propagation time from 4 months to approx. 35 days making it comparable to the time taken in conventional nursery practice. Plantlet regeneration at low I A A concentration in one step and without vitrification o f foliage will also make it economically feasible.
_J
x
I,,
o o I
5O
no u_
L.b Z O z o n
z tn
25
LIJ rr
Acknowledgements Authors are grateful to Dr. P.L. G a u t a m (Joint Director), Hill Campus, Ranichauri for guidance in the present investigation. References
o 1 LIGHT
2 3 4 INTENSITY (K LUX)
Fig. 2. Influenceof light intensity (K lux) on days taken for shoot proliferation (El) and % responding explants (121)after 25 days of incubation on MS medium supplemented with 1 mg 1-I IAA. Mean + S.E. of three replications with 40 samples in each treatment.
1 B.W.M. Grout, Cauliflower (Brassica oleracea var. botrytis L.), in: Y.P.S. Bajaj (Ed.), Biotechnology in Agriculture and Forestry Crops II, Vol. 6, Springer Verlag, 1988, pp. 211-225. 2 S.Y. Ze and B.B. Johnson, Cole Crops, in: P.V. Ammirato, D.A. Evans, W.R. Sharp and Y. Yamada (Eds.), Handbook of Plant Cell Culture Crop Species, Vol. 3, Macmillan, lnc, New York, London, 1984, pp. 227-246.
178 3
4
5
6
7
8
V.A. Kumar, A. Kumar and J. Kumar, In vitro plant regeneration of cauliflower (Brassica oleracea convar botrytis var botrytis) from mature curd. Indian J. Agric. Sci., 62 (1992) 429-431. D.G.A. Walkey and J.G.M. Woolfit, Rapid clonal multiplication of cauliflower by shake culture. J. Hort. Sci., 45 (1970) 205-206. B.W.M. Grout and P. Crisp, Practical aspects of the propagation of cauliflower by meristem culture. Acta Hort., 78 (1977) 289-296. K.C. Short, J. Warburton and A.V. Roberts, In vitro hardening of cultured cauliflower and chrysanthemum plantlets to humidity. Acta Hort., 212 (1987) 329-334. T. Murashige and F A. Skoog, A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant., 15 (1962) 473-497. D.R. Hoagland, Lectures on the Inorganic Nutrition of
Plants, Chronica Botanica Company, Waltham, Mass. E.U. Linsmair and F.A. Skoog, Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18 (1965) 100-127. 10 P. Crisp and D.G.A. Walkey, The use of aseptic meristem culture in cauliflower breeding. Euphytica, 23 (1974) 305-313. 11 M. Buiatti, S. Baroncelli, A. Bennici, M. Pagliai and R. Tesi, Genetics of growth and differentiation in vitro of Brassica oleracea var. botrytis. II. An in vitro and in vivo analysis of a diallel cross. Z. Pflanzenphysiol., 72 (1974) 269-274. 12 M. Buiatti, S. Baroneelli and A. Bennici, Genetics of growth and differentiation in vitro of Brassica oleracea var. botrytis. IV. Genotype-hormone interactions. Z. Pflanzenphysiol., 73 (1974) 298-302. 9