Plant Cell Reports (1984) 3:1-4
Plant Cell Reports © Springer-Verlag 1984
Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson S. Singh and N. Chandra Department.of Botany, University ofRajasthan, Jaipur-302 004, India Received August 1, 1983 / Revised version received November 16, 1983 - Communica}ed by J. Widholm
Abstract Prolific shoot bud differeDtiation was induced in callus and suspension cultures of hypocotyl origin in B__~rassica campestris cv. Yellow Sarson on MS medium supplemented with K - - ~ - ~ ' ~ , ~ p M ) or BA (1),~-22ol ~M), Plan.tlets were obtained by roo%ing the in vitro differentiated shoots, Histological studies revealed a unique mode of meristemoid formation, Abbreviations MS 2,4-9 BA IAA IBA K
NAA
: : : : : : :
Murash~ge a~d Skoog (1962) 2,4-dichlorophenoxyacetic acid Benzyladenine Indole-3-acetic acid Indolebutyric acid Kinetin Naphthalene acetic acid.
Introduction We have undertaken a detailed study of factors affecting differentiation in explants, callus and suspension cultures of different Brass]ca species (Pareek s~d Chandra. 1978, 1981; Singh e~t a_~lo 1981). The main object of the present study was to determine the effects of different auxins and cytokinins and their combinatior~ on growth and differentiation of callus and suspension cultures of Brassica cam~estris cv. Yellow Samson, an important vegetable oil crop widely grown in India. ~lantlet formation through anther culture of ~. has been reported earlier e~t a~l, 1975; Keller and Armstrong, 1979). Schenk and Hoffmann (1979) reported rhizogenesis in callus cultures of protoplast origin. Bhattacharya and Sen (1980) obtained plantlets through somatic embryogenesis in ~. c _ ~ e s t r i s vat. toria. Material and Methods Seeds of Brassica campestris cv. Yellow ~arso~ were procured from the Indian Agricultural Research Institute, New Delhi. They were grown aseptically after surface
sterilization in 0.1% mercuric chloride and thorough washing with sterile distilled water. Hypocotyl segments (ca 10 ~am long) taken from 15 d old plants were cultured on agarised MS medium (Murashige and Skoog, 1962) supplemented with various growth substances. Though callus was initiated on several concentrations but best callus formation was recorded on MS medium with 2.3 ~ M K + 0.57 ~ M IAA. Callus was multiplied on the same medium by subculturing (monthly) through 6-8 passages before starting experiments.Oae piece of callus (ca 200 mg fresh wt.) was transferred per flask and oOservations were recorded in 30 d of incubation. Five replicates of each treatment were kept for observation and experiments were repeated twice. Standard error of the arithmetic mean was calculated by the formula given by Snedecor (1956). Suspension cultures were initiated by transferring callus to MS liquid medium of the same composition and maintained on a gyratory shaker (Emenvee. Poona) at 100 rpm. The pH of the medium was adjusted to 5.8 with 0.1N ~C1/NaOH before autoclaving at 1.05 kg/cm for 15 min. All the experiments on callus and suspension were incubated in a growth room at 26 ! 2oc under continuous light of 3000 lux from fluorescent tubes and incandescent bulbs. For histological preparations, small pieces of calli were fixed in Formalineacetic acid - alcohol (5:5:90) at weekly intervals after transfer to the induction me d ium (MS + 22.1 ~ M BA). Dehydration was done in tertiary butyl alcohol-xylol series (Johansen, 1940). Sections were cut at I 0 - 1 2 p m thickness and stained with safr~min and light green. Results and Discussion Differentiation in callus cultures : Callus f hyPocotyl origin grown on MS + IAA 0.57 ~M) ÷ K (2.3 ~M) was fragile and unorganized. In the presence of auxin alone (IAA, IBA, NAA, 2,4-D), better growth (fresh weight yield) was obtained at all different levels tried in comparison to
~
2 to controls (MS medium without any growth substance') where growth was negligible and callus necrosed after 15-20 d of incubation. Among the auxins tried singly, best growth was obtained with 5.7~aM IAA. On higher levels of any auxin, ~ rooting was observed. The best rooting response was obtained on IBA (24.6 ~ M ) and roots were longer, thicker and with laterals. In the presence of NAA roots were smaller but numerous. 2,4-~ proved to be a poor root inducer. Amongst the two cytokinins, K and BA, used singly, the growth of callus was better on lower levels of K (2.3 ~ M ) and declined with increasing levels. The growth was poor on BA supplemented media. Shoot buds differentiated in the third week of incubation, in response to both K and BA and their number increased with increasing levels of the cytokinin. BA (22.1 ~M) induced the maximum number of shoot buds (Fig. IA; Table-l). Table I. Effects of K and BA added singly to MS medium on growth and differentiation in hypocotyl callus of Brassica campestris cv. Yellow Sarson. MS $ K/BA (~M)
Fresh weight (g/flask)
Percentage cultures with differentiating shoots
K
2.3 4.6 13.9 23.2
4.27 3.23 2.28 1.95
~ 0.21 • 0.44 ± 0.18 -~ 0.23
NR 40 40 100
BA
2.2 4.4 13.I 22.1
3.18 2.57 1.93 1.16
+_ 0.32 • 0.08 +_ 0.27 _+ 0.17
NR 20 100 100
Figures in second column represent mean values + standard error. NR = No response. The growth of callus, in general, was found to be better on a combination of an auxin and a cytokinin. The maximum fresh weight of callus was obtained with a combination of K ( 1 3 . 9 ~ M ) + IBA (14.7taM). A general trend of improved growth yield with increasing auxin level was noted. Better growth yield, in most cases, was found at higher levels of an auxin in various combinations of IAA (2.8-17.1 ~M), IBA (2.5 ~M-14.7 ~M) or NAA (2.7-16.1 ~M) with relatively higher levels of K (]3.9-23.2 ~M) or BA (13.3-22.1 ~M). On all the combinations with lower level of a cytokinin (K, 4.6 ~M; BA, 4.4~M), shoot buds were formed in a very low percentage of cultures. Although numerous shoot buds were formed in combinations with higher levels of K/BA, only in a few cases did they exceed the number obtained in controls (cytokinin alone). There was no shoot bud differentiation in any of the
combination with higher level of an auxin except in the medium supplemented with 22.1 ~ M BA where shoot buds were induced in a low percentage of cultures. 2,4-~ suppressed shoot bud induction and only a few shoot buds differentiated in combinations of higher levels of a cytokinin with lower levels of 2,4-D. Differentiation in Suspension cultures : A suspension was obtained on MS medium with IAA (0.57 ~M) ~ K (2.3 taM) after the second subculture. Several concentrations of an auxin (IAA/IBA), a cytokinin (K/BA) and their combinations were tested to see their comparative morphogenic effects. In the presence of IAA (2.8-5.7 ~M) or IBA (2.54.9 I~M) there were a large number of single cells. On increasing the IAA/IBA level, the number of single cells declined and number and size of cell clumps increased. On lower levels of IAA (5.7 ~M) or IBA (4.9 taM) thin and white roots were formed and their number increased with increasing levels of the auxin. Maximal rooting res~ ponse was observed on 24.6 paM IBA where roots were longer and culture flasks were completely filled with such roots within 30 d of incubation. When suspension cultures grown on IAA (0.57 ~M) + K (2.3 ~M) were transferred to IAA free medium the number of cell clumps increased and they turned green and enlarged in size. On higher K level (4.6-23.2 FaM), numerous structures each having a spherical callus mass and a single root were formed (Fig. IB). Shoot buds were induced at all levels of K tried (Fig. IC) but their number increased at higher levels (13,923.2 ~ M ) . The response on BA supplemented media was similar to that noted on K supplemented media. In the present study shoot bud differentiation in B. campestris was fotuqd to occur profusely on MS medium with a cytokinin (K, 23.2 ~ M or BA, 22.1 ~M). ])unwell (1981) found B. c ~ e s t r i s to be the least amenable in a comparative study of regeneration of shoot buds in leaf discs of three species of Brassica. In his studies, regeneration of shoot buds was found to occur in B. campestris in the presence of 4.4-~M BA and 53.7 ~ M NAA, in B. oleraoea var. in the presence of 44.4 ~M BA and 5.4 ~M NAA and in B. ~ in a combination of 44.4 ~M BA and ~3.7 ~ M of NAA. From his studies sit is clear that comparatively higher levels cf NAA along with a lower level of BA were required for optimal differentiation of leaf discs of B. campestris However, in B. c~__~stri~ h y p o c o t y i ~ e g m e n t s (Singh e't al, 1981) ~ in the present study of the callus, shoot bud differentiation occurred on MS medium with K (4.6-23.2 ~M) + NAA (2.75.4 ~M) and with BA (22.1 ~uM)/K (23.2 ~M~ respectively. Thus regeneration of shoot buds in different explants and callus of B. campestr!s has been found to occur in response to different hormonal balances. This has been explained as due to the physiological state of the explant (Dunwell, 1976) or as controlled by the genotype (Buiattl et al, 1974).
2,4-9 was inhibitory for shoot bud induction in callus as well as suspension cultures of B. camoestris, although it promoted callus growth. In Brassica oleracea also, 2,4-D has been reported to be inhibitory for shoot bud induction as in cauliflower (M-~rgara, 1969) :~r~d i~ marrow stem kale (Lustinec and Horak, 1970).
(Fig. ID). However, in combinations of an auxin and a cytokinin callus developed first followed by root formation. H i s t o l o ~ of Differentiation : The callus grown on an induction medium and periodically fixed and se~tioned revealed ~ series of changes leading to organized growth. The unorganized callus showed loosely arranged cells full of starch grains and varying in size and shape. The callus cultured for one week did not show any significant change but in the second week it becs~e compact and showed smaller densely packed cells without intercellular spaces.
A Fig. 1(A-D) Differentiation and plant!et formation in callus and suspension cultures of Brassica campestris cv. Yellow Sarson. A
:
Differentiation of shoot buds in 30d old callus cultures on MS * BA (22.1 ~M).
B
:
Peculiar structures, each with a single root and a callus mass, differentiated in 30 d old suspension cultures on MS * K (23.2 ~ M ) .
C
:
Shoot buds from 30 d old suspension cultures growing on MS + BA (22.1 DM).
D
:
A plantlet formed by rooting of shoot on a filter paper bridge in MS ÷ NAA (26.5 ~M).
Rooting of differentiated shoots : Shoots (3-4 cm long) differentiated in vitro from callus cultures were cut from the base by a sharp scalpel sm.d transferred aseptically to filter paper bridges in wide mouth (25 mm diameter) culture tubes containing 10 ml MS liquid medium. The level of the medium was Kept well below the filter paper bridge. When isolated shoots were cultured on MS ÷ IAA (17.1 ~M), rooting occurred in 10-15 d in 60% cultures while in others callus was formed. On higher levels of IAA (28.5 DM) rooting was observed in 100% cultures. Response in terms of number and length of roots was better on MS ÷ IBA (24.6 pM). In many cases, roots differentiated directly from the base of the shoots
Fig. 2(A-D) : Histology of differentiation of meristemoids and shoot buds in callus cultures of Brassica campestris cv. Yellow Sarson. A
B
:
:
C,D:
Section through differentiating callus showing internal divisions a cell X 480.
in
Sections passing longitudinally through a developing bipolar meristemoid. Note lower portion embedded in the parent callus tissue. X 180. Sections through a meristemoid showing developing shoot bud primordia on its outer surface X 180 and a shoot bud X 120 respectively.
Meristemoids (meristematic centres) were initiated in the sub-surface layers (4-5 cell deep) in callus. There is evidence that meristemoids originated in single cells as in many cases several daughter cells were seen inside the initial cell, as if they were formed by divisions and subdivisions of the same cell (Fig. 2A). Such a mode of meristemoid initiation has also been noted recently in Nicotiana glauca (Nuti-Ronchi, 1981). The resulting cells were smaller with dense cytoplasmic contents and conspicuous nuclei. To begin with, the meristematic activity in these cell masses was diffused but later on it was restricted to the two opposite poles one in the outer part and the other in the tip of the inner embedded part which showed limited meristematic activity and resembled the root pole of an embryoid. Such bipolar meristemoids resembled arrested embryoids and showed a limiting layer of densely cytoplasmic cells (Fig. 2B). Eventually, the outer end expanded by cell proliferation and projected out of the callus mass (Fig. 2C). On the outer facet of this expanded portion of the meristemoid several shoot-buds developed, each with a shoot apex flanked by leaf primordia (Fig. 2D). Quite commonly, new shoot buds developed from various parts of differentiated shoots.
References Bhattacharya NM, Sen SK (1980) Z Pflanzenphysiol 99 : 357-365 Buiatti M, Baroncelli S, Bennici A (1974) Z Pflanzenzuchtg 73:298-302 Dunwell JM (7976) Env Exp Bot 16:~09-118
In the present study some peculiar structures, each having a callus mass and a single root were formed in large numbers. Although the nature of such structures could not be ascertained, they might have originated either by callusing of the shoot pole of an embryoid or by formation of callus in a detached root or by formation of single root in a callus aggregate.
Nuti-Ronchi V (1981) Can J Bot 59:1969-1977
Acknowledgement Thanks are due to the Council of Scientific and Industrial Research, New Delhi for financial assistance.
Dunwell JM (1981) J Exp Bot 32:789-799 Johansen hA (1940) Plant Microtechnique. Mc Graw Hill Co, New York Keller WA, Armstrong KC (1979) Theor Appl Genet 55:65-67 Keller WA, Rajhatny T, Lacapra J (1975) Can J Genet Cytol 17:655-666 Lustinec J, Horak J (1970) Experientia 26:919-920 Margara J (1969) Ann Physiol veg Paris 11:95-112 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Pareek LK, Chandra N (1978) Plant Sci Lett 11:311-316 Pareek LK, Chandra N (1981) Indian J ~xp Biol 19:874-875
Schenk HR, Hoffmann F (1979) Z Pflanzenzuchtg 82:354-360 Singh S, Banu S, Pareek LK, Chandra N (1981) Indian J Exp Biol 19:658-660 Snedecor GW (1956) Statistical methods applied to experiments in agriculture and biology. Iowa State College Press, Ames.
Plant Cell Reports © Springer-Verlag 1984
Plant regeneration in callus and suspension cultures of Brassica campestris cv. Yellow Sarson S. Singh and N. Chandra Department.of Botany, University ofRajasthan, Jaipur-302 004, India Received August 1, 1983 / Revised version received November 16, 1983 - Communica}ed by J. Widholm
Abstract Prolific shoot bud differeDtiation was induced in callus and suspension cultures of hypocotyl origin in B__~rassica campestris cv. Yellow Sarson on MS medium supplemented with K - - ~ - ~ ' ~ , ~ p M ) or BA (1),~-22ol ~M), Plan.tlets were obtained by roo%ing the in vitro differentiated shoots, Histological studies revealed a unique mode of meristemoid formation, Abbreviations MS 2,4-9 BA IAA IBA K
NAA
: : : : : : :
Murash~ge a~d Skoog (1962) 2,4-dichlorophenoxyacetic acid Benzyladenine Indole-3-acetic acid Indolebutyric acid Kinetin Naphthalene acetic acid.
Introduction We have undertaken a detailed study of factors affecting differentiation in explants, callus and suspension cultures of different Brass]ca species (Pareek s~d Chandra. 1978, 1981; Singh e~t a_~lo 1981). The main object of the present study was to determine the effects of different auxins and cytokinins and their combinatior~ on growth and differentiation of callus and suspension cultures of Brassica cam~estris cv. Yellow Samson, an important vegetable oil crop widely grown in India. ~lantlet formation through anther culture of ~. has been reported earlier e~t a~l, 1975; Keller and Armstrong, 1979). Schenk and Hoffmann (1979) reported rhizogenesis in callus cultures of protoplast origin. Bhattacharya and Sen (1980) obtained plantlets through somatic embryogenesis in ~. c _ ~ e s t r i s vat. toria. Material and Methods Seeds of Brassica campestris cv. Yellow ~arso~ were procured from the Indian Agricultural Research Institute, New Delhi. They were grown aseptically after surface
sterilization in 0.1% mercuric chloride and thorough washing with sterile distilled water. Hypocotyl segments (ca 10 ~am long) taken from 15 d old plants were cultured on agarised MS medium (Murashige and Skoog, 1962) supplemented with various growth substances. Though callus was initiated on several concentrations but best callus formation was recorded on MS medium with 2.3 ~ M K + 0.57 ~ M IAA. Callus was multiplied on the same medium by subculturing (monthly) through 6-8 passages before starting experiments.Oae piece of callus (ca 200 mg fresh wt.) was transferred per flask and oOservations were recorded in 30 d of incubation. Five replicates of each treatment were kept for observation and experiments were repeated twice. Standard error of the arithmetic mean was calculated by the formula given by Snedecor (1956). Suspension cultures were initiated by transferring callus to MS liquid medium of the same composition and maintained on a gyratory shaker (Emenvee. Poona) at 100 rpm. The pH of the medium was adjusted to 5.8 with 0.1N ~C1/NaOH before autoclaving at 1.05 kg/cm for 15 min. All the experiments on callus and suspension were incubated in a growth room at 26 ! 2oc under continuous light of 3000 lux from fluorescent tubes and incandescent bulbs. For histological preparations, small pieces of calli were fixed in Formalineacetic acid - alcohol (5:5:90) at weekly intervals after transfer to the induction me d ium (MS + 22.1 ~ M BA). Dehydration was done in tertiary butyl alcohol-xylol series (Johansen, 1940). Sections were cut at I 0 - 1 2 p m thickness and stained with safr~min and light green. Results and Discussion Differentiation in callus cultures : Callus f hyPocotyl origin grown on MS + IAA 0.57 ~M) ÷ K (2.3 ~M) was fragile and unorganized. In the presence of auxin alone (IAA, IBA, NAA, 2,4-D), better growth (fresh weight yield) was obtained at all different levels tried in comparison to
~
2 to controls (MS medium without any growth substance') where growth was negligible and callus necrosed after 15-20 d of incubation. Among the auxins tried singly, best growth was obtained with 5.7~aM IAA. On higher levels of any auxin, ~ rooting was observed. The best rooting response was obtained on IBA (24.6 ~ M ) and roots were longer, thicker and with laterals. In the presence of NAA roots were smaller but numerous. 2,4-~ proved to be a poor root inducer. Amongst the two cytokinins, K and BA, used singly, the growth of callus was better on lower levels of K (2.3 ~ M ) and declined with increasing levels. The growth was poor on BA supplemented media. Shoot buds differentiated in the third week of incubation, in response to both K and BA and their number increased with increasing levels of the cytokinin. BA (22.1 ~M) induced the maximum number of shoot buds (Fig. IA; Table-l). Table I. Effects of K and BA added singly to MS medium on growth and differentiation in hypocotyl callus of Brassica campestris cv. Yellow Sarson. MS $ K/BA (~M)
Fresh weight (g/flask)
Percentage cultures with differentiating shoots
K
2.3 4.6 13.9 23.2
4.27 3.23 2.28 1.95
~ 0.21 • 0.44 ± 0.18 -~ 0.23
NR 40 40 100
BA
2.2 4.4 13.I 22.1
3.18 2.57 1.93 1.16
+_ 0.32 • 0.08 +_ 0.27 _+ 0.17
NR 20 100 100
Figures in second column represent mean values + standard error. NR = No response. The growth of callus, in general, was found to be better on a combination of an auxin and a cytokinin. The maximum fresh weight of callus was obtained with a combination of K ( 1 3 . 9 ~ M ) + IBA (14.7taM). A general trend of improved growth yield with increasing auxin level was noted. Better growth yield, in most cases, was found at higher levels of an auxin in various combinations of IAA (2.8-17.1 ~M), IBA (2.5 ~M-14.7 ~M) or NAA (2.7-16.1 ~M) with relatively higher levels of K (]3.9-23.2 ~M) or BA (13.3-22.1 ~M). On all the combinations with lower level of a cytokinin (K, 4.6 ~M; BA, 4.4~M), shoot buds were formed in a very low percentage of cultures. Although numerous shoot buds were formed in combinations with higher levels of K/BA, only in a few cases did they exceed the number obtained in controls (cytokinin alone). There was no shoot bud differentiation in any of the
combination with higher level of an auxin except in the medium supplemented with 22.1 ~ M BA where shoot buds were induced in a low percentage of cultures. 2,4-~ suppressed shoot bud induction and only a few shoot buds differentiated in combinations of higher levels of a cytokinin with lower levels of 2,4-D. Differentiation in Suspension cultures : A suspension was obtained on MS medium with IAA (0.57 ~M) ~ K (2.3 taM) after the second subculture. Several concentrations of an auxin (IAA/IBA), a cytokinin (K/BA) and their combinations were tested to see their comparative morphogenic effects. In the presence of IAA (2.8-5.7 ~M) or IBA (2.54.9 I~M) there were a large number of single cells. On increasing the IAA/IBA level, the number of single cells declined and number and size of cell clumps increased. On lower levels of IAA (5.7 ~M) or IBA (4.9 taM) thin and white roots were formed and their number increased with increasing levels of the auxin. Maximal rooting res~ ponse was observed on 24.6 paM IBA where roots were longer and culture flasks were completely filled with such roots within 30 d of incubation. When suspension cultures grown on IAA (0.57 ~M) + K (2.3 ~M) were transferred to IAA free medium the number of cell clumps increased and they turned green and enlarged in size. On higher K level (4.6-23.2 FaM), numerous structures each having a spherical callus mass and a single root were formed (Fig. IB). Shoot buds were induced at all levels of K tried (Fig. IC) but their number increased at higher levels (13,923.2 ~ M ) . The response on BA supplemented media was similar to that noted on K supplemented media. In the present study shoot bud differentiation in B. campestris was fotuqd to occur profusely on MS medium with a cytokinin (K, 23.2 ~ M or BA, 22.1 ~M). ])unwell (1981) found B. c ~ e s t r i s to be the least amenable in a comparative study of regeneration of shoot buds in leaf discs of three species of Brassica. In his studies, regeneration of shoot buds was found to occur in B. campestris in the presence of 4.4-~M BA and 53.7 ~ M NAA, in B. oleraoea var. in the presence of 44.4 ~M BA and 5.4 ~M NAA and in B. ~ in a combination of 44.4 ~M BA and ~3.7 ~ M of NAA. From his studies sit is clear that comparatively higher levels cf NAA along with a lower level of BA were required for optimal differentiation of leaf discs of B. campestris However, in B. c~__~stri~ h y p o c o t y i ~ e g m e n t s (Singh e't al, 1981) ~ in the present study of the callus, shoot bud differentiation occurred on MS medium with K (4.6-23.2 ~M) + NAA (2.75.4 ~M) and with BA (22.1 ~uM)/K (23.2 ~M~ respectively. Thus regeneration of shoot buds in different explants and callus of B. campestr!s has been found to occur in response to different hormonal balances. This has been explained as due to the physiological state of the explant (Dunwell, 1976) or as controlled by the genotype (Buiattl et al, 1974).
2,4-9 was inhibitory for shoot bud induction in callus as well as suspension cultures of B. camoestris, although it promoted callus growth. In Brassica oleracea also, 2,4-D has been reported to be inhibitory for shoot bud induction as in cauliflower (M-~rgara, 1969) :~r~d i~ marrow stem kale (Lustinec and Horak, 1970).
(Fig. ID). However, in combinations of an auxin and a cytokinin callus developed first followed by root formation. H i s t o l o ~ of Differentiation : The callus grown on an induction medium and periodically fixed and se~tioned revealed ~ series of changes leading to organized growth. The unorganized callus showed loosely arranged cells full of starch grains and varying in size and shape. The callus cultured for one week did not show any significant change but in the second week it becs~e compact and showed smaller densely packed cells without intercellular spaces.
A Fig. 1(A-D) Differentiation and plant!et formation in callus and suspension cultures of Brassica campestris cv. Yellow Sarson. A
:
Differentiation of shoot buds in 30d old callus cultures on MS * BA (22.1 ~M).
B
:
Peculiar structures, each with a single root and a callus mass, differentiated in 30 d old suspension cultures on MS * K (23.2 ~ M ) .
C
:
Shoot buds from 30 d old suspension cultures growing on MS + BA (22.1 DM).
D
:
A plantlet formed by rooting of shoot on a filter paper bridge in MS ÷ NAA (26.5 ~M).
Rooting of differentiated shoots : Shoots (3-4 cm long) differentiated in vitro from callus cultures were cut from the base by a sharp scalpel sm.d transferred aseptically to filter paper bridges in wide mouth (25 mm diameter) culture tubes containing 10 ml MS liquid medium. The level of the medium was Kept well below the filter paper bridge. When isolated shoots were cultured on MS ÷ IAA (17.1 ~M), rooting occurred in 10-15 d in 60% cultures while in others callus was formed. On higher levels of IAA (28.5 DM) rooting was observed in 100% cultures. Response in terms of number and length of roots was better on MS ÷ IBA (24.6 pM). In many cases, roots differentiated directly from the base of the shoots
Fig. 2(A-D) : Histology of differentiation of meristemoids and shoot buds in callus cultures of Brassica campestris cv. Yellow Sarson. A
B
:
:
C,D:
Section through differentiating callus showing internal divisions a cell X 480.
in
Sections passing longitudinally through a developing bipolar meristemoid. Note lower portion embedded in the parent callus tissue. X 180. Sections through a meristemoid showing developing shoot bud primordia on its outer surface X 180 and a shoot bud X 120 respectively.
Meristemoids (meristematic centres) were initiated in the sub-surface layers (4-5 cell deep) in callus. There is evidence that meristemoids originated in single cells as in many cases several daughter cells were seen inside the initial cell, as if they were formed by divisions and subdivisions of the same cell (Fig. 2A). Such a mode of meristemoid initiation has also been noted recently in Nicotiana glauca (Nuti-Ronchi, 1981). The resulting cells were smaller with dense cytoplasmic contents and conspicuous nuclei. To begin with, the meristematic activity in these cell masses was diffused but later on it was restricted to the two opposite poles one in the outer part and the other in the tip of the inner embedded part which showed limited meristematic activity and resembled the root pole of an embryoid. Such bipolar meristemoids resembled arrested embryoids and showed a limiting layer of densely cytoplasmic cells (Fig. 2B). Eventually, the outer end expanded by cell proliferation and projected out of the callus mass (Fig. 2C). On the outer facet of this expanded portion of the meristemoid several shoot-buds developed, each with a shoot apex flanked by leaf primordia (Fig. 2D). Quite commonly, new shoot buds developed from various parts of differentiated shoots.
References Bhattacharya NM, Sen SK (1980) Z Pflanzenphysiol 99 : 357-365 Buiatti M, Baroncelli S, Bennici A (1974) Z Pflanzenzuchtg 73:298-302 Dunwell JM (7976) Env Exp Bot 16:~09-118
In the present study some peculiar structures, each having a callus mass and a single root were formed in large numbers. Although the nature of such structures could not be ascertained, they might have originated either by callusing of the shoot pole of an embryoid or by formation of callus in a detached root or by formation of single root in a callus aggregate.
Nuti-Ronchi V (1981) Can J Bot 59:1969-1977
Acknowledgement Thanks are due to the Council of Scientific and Industrial Research, New Delhi for financial assistance.
Dunwell JM (1981) J Exp Bot 32:789-799 Johansen hA (1940) Plant Microtechnique. Mc Graw Hill Co, New York Keller WA, Armstrong KC (1979) Theor Appl Genet 55:65-67 Keller WA, Rajhatny T, Lacapra J (1975) Can J Genet Cytol 17:655-666 Lustinec J, Horak J (1970) Experientia 26:919-920 Margara J (1969) Ann Physiol veg Paris 11:95-112 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Pareek LK, Chandra N (1978) Plant Sci Lett 11:311-316 Pareek LK, Chandra N (1981) Indian J ~xp Biol 19:874-875
Schenk HR, Hoffmann F (1979) Z Pflanzenzuchtg 82:354-360 Singh S, Banu S, Pareek LK, Chandra N (1981) Indian J Exp Biol 19:658-660 Snedecor GW (1956) Statistical methods applied to experiments in agriculture and biology. Iowa State College Press, Ames.
