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Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Saskatchewan S7N OW9 Canada
Effect of Growth Regulators on Plant Regeneration from Shoot Apical Meristems of Cassava (Manihot esculenta CRANTZ) and on the Culture of Internodes in vitrol)
N. G. NAIR2), K. K. KARTHA, and o. L. GAMBORG
With 1 figure
Received April 24, 1979 . Accepted May 12, 1979
Summary
The regeneration of shoots from cassava meristems was described as a function of presence or absence of cytokinins, NAA and GA3. Out of 4 cytokinins, BA was best suited for plant regeneration followed by zeatin, kinetin and 2iP. In conjunction with either 1.0 or 0.1 ,uM NAA and O.l,uM GA3, all levels of BA induced plant regeneration in 100 % of the meristems. Shoot development was poor in most combinations involving higher concentrations of NAA. Addition of GA3 improved plant regeneration and its effect was more pronounced at higher concentrations of NAA. Attempts to induce shoot regeneration from callus derived from internodal segments were unsuccessful.
Key words: Cytokinins, auxins, cassava mosaic disease, plant regeneration, meristem culture, differentiation, regeneration index, internode culture.
Introduction
Cassava (Manihot esculenta CRANTZ), a vegetatively propagated tuber crop, is one of the 12 to 15 major crops of the world. Its ability to yield well even under marginal farming conditions and to produce more food energy per unit area as compared to most other crops make it a valuable source of food in India and some of the African and South American countries.
KARTHA et al. (1974) developed a technique for the culture and plant regeneration from the shoot apical meristems of cassava. Employing that technique they (KARTHA and GAMBORG, 1975) later produced cassava plants free of mosaic disease.
Abbreviations: NAA, naphthalene acetic acid; BA, benzyladenine; GA3, gibberellic acid; 2iP, isopentenyl adenine.
1) NRCC No. 17573. 2) Visiting Scientist, Central Tuber Crops Research Institute, Trivandrum, Kerala State,
India.
z. Pflanzenphysiol. Bd. 95. S. 51-56. 1979.
52 N. G. NAIR, K. K. KARTHA, and O. 1. GAMBORG
Their meristem culture technique together with the low rate of insect transmiSSion of the mosaic disease (NAIR, unpublished) form an ideal and useful tool for the regeneration and rehabilitation of promising varieties, some of which have already gone out of cultivation due to this disease.
The present work was undertaken in an attempt to elucidate the in vitro response of the shoot apical meristems and internodal segments of two important Indian cassava cultivars to various levels of growth hormones under nutritionally and environmentally defined conditions. Results of the experiments on the interaction of 4 cytokinins with NAA in the presence or absence of GAs in inducing plant regeneration and also on the vigor of the regenerated plants are presented. An attempt has also been made to induce shoot regeneration from young internodal segments.
Materials and Methods
Dormant cuttings of the cassava variety «M 4» obtained from the Central Tuber Crops Research Institute, Trivandrum, India, were used in meristem culture experiments while «M 4» and variety «Kalikalan» (obtained from the same source) were used in the internode culture. Dormant stakes were cut into 10 cm sections, the upper cut ends sealed with paraffin, and potted in a mixture of vermiculite, peat moss, and sand (3 : 2 : 1). The material was grown at 26°C 16 hi day photoperiods (8000 lux intensity from combined fluorescent and incandescent lamps) and 70 Ofo relative humidity.
Shoot-tips developing from these stakes were used as source of meristems. Shoots developing from the axial buds were also used. Apical meristems of 0.2 to 0.4 mm in length were aseptically isolated and cultured according to the procedure reported earlier (KARTHA et aI., 1974). In the present experiments, 4 cytokinins, viz. zeatin (trans 6-(4-hydroxy-3-methylbut-2-enyl)-aminopurine) 2iP [6(y ,y-dimethylallylamino )-purine], kinetin (6-furfurylaminopurine) and BA (6-benzylaminopurine) were tested in 10.0, 1.0, 0.5 and O.l,uM levels in combinations with 3 levels of NAA (10.0, 1.0, and 0.1 ,uM) in the presence or absence of O.l,uM GA3• We tested 24 hormonal combinations for each cytokinin and auxin level. Ten meristems were cultured under each set of conditions.
Internodal segments from the cassava cultivars «M4» and «Kalikalan» were disinfected for 10 min in 20 Ofo commercial bleach (1.2 % sodium hypochlorite), thoroughly washed 4 times with autoclaved distilled water and cut into segments of 5 mm in length. Each internodal segment was grown in MS medium (MURASHIGE and SKOOG, 1962) solidified with 0.8 Ofo agar and supplemented with 0.5 ,uM BA, 1.0 ,uM NAA and 0.1 ,uM GA3 • The resulting callus was subsequently transferred to medium devoid of GA3 (TILQUIN, 1978).
Both meristem-tips and internodal segments were cultured under 26°C, 16 hi day photoperiods (4000 lux from fluorescent and incandescent lamps) and 70 Ofo relative humidity.
Results and Discussion
The results on the effect of various combinations of the 4 cytokinins and NAA in the presence or absence of GA3 in inducing growth and plant regeneration are presented in Fig. 1. Since roots could be readily induced in shoots, all the differentiated shoots with or without concomitant roots were classified as plant regeneration. Depending upon the hormone concentration, the callus may be with or without roots.
Z. PJlanzenphysiol. Bd. 95. S. 51-56. 1979.
Cassava meristem and internode culture 53
~']III I I Ii w u a:
1'"1 o w a:
ABeD abed ABeD abed ABeD abed ABeD abed ZEATIN 2iP KINETIN BENZYLADENINE
• PLANT REGENERAT ION G CALLUS ONLY D WITHOUT GROWTH
Fig. 1: Effect of 4 cytokinins and NAA in inducing plant regeneration from the cassava meristems when cultured in the presence or absence of GA3• ABeD represents cytokinin concentrations of 10.0, 1.0, 0.5 and 0.1 ,uM, respectively, in the presence of 0.1 ,uM GAs while abcd denotes identical concentrations of cytokinins in the absence of GAs.
A general trend emerges from the experiments: in all combinations, BA enhanced plant regeneration more than zeatin, kinetin and 2iP. Shoot development was poor in most of the combinations involving 10.0.uM NAA.
In the presence of 0.1 .uM GAs and when combined with 10.0.uM each of NAA and zeatin and 0.5 or 0.1 .uM of BA, 100 % plant regeneration was achieved. More than 50 Ofo of isolated meristems differentiated into plantlets when grown in the presence of 1.0.uM zeatin, 10.0.uM 2iP, O.S.uM kinetin or 1.0.uM BA combined with 10.0 or O.l.uM NAA and O.l.uM GA3• When GAs was omitted from the medium, cytokinin combinations with 10.0.uM NAA generally induced less than 50 Ofo plant regeneration; the least effective being 2iP and kinetin.
One hundred per cent plant regeneration was obtained at all levels of BA in combination with 1.0.uM NAA when GA3 was present. Even in the absence of GA3, BA supported 100 Ofo plant regeneration in all combinations except 0,1 .uM.
z. Pflanzenphysiol. Bd. 95. S. 51-56. 1979.
54 N. G. NAIR, K. K. KARTHA, and O. L. GAMBORG
The vigor of the regenerated shoots and roots was recorded on a scale of 1 to 5 on the basis of visual assessment (Table 1). Shoots with or without roots of grade 5,4
and 3 on transfer to rooting medium (MS + 1.0 pM NAA + 0.5 f-lm BA) produced healthy and vigorous plants whereas those of grade 2 and 1 did not. Often material of these categories remained stunted and did not produce roots even after one month of culture. Moreover, material of grade 1 and 2 when supplied with high levels of NAA, produced extensive basal callus. On transfer to GA-free medium, although roots were induced, the callus growth predominated the shoot development resulting in stunted growth. Higher concentrations of NAA induced more vigorous root formation. Under low concentrations a concomitant reduction in the number of roots was observed. It was again evident that NAA at 0.1 f-lM with all levels of BA was best suited in inducing 100 Ofo plant regeneration. In an earlier study KARTHA et al. (1974) found that 1.0 pM was the more suitable concentration. The difference in
Table 1: Effect of growth regulators on the vigor of shoots regenerated from cassava meristems and on root formation.
Hormone levels CuM)
NAA
Zeatin 10.0
1.0 0.5 0.1
liP
10.0 1.0 0.5 0.1
Kinetin 10.0
1.0 0.5 0.1
BA 10.0
1.0 0.5 0.1
Plant vigora)
10.0 1.0 0.1
4 2 1 1
1 1 1 o
5 1 1
2 4 5 2
5 2 1 1
1 1 1 o
o 1 1 1
5 4 3 2
5 5 3 2
3 1 1 2
1 2 2 1
3 5 5 4
without GAa
10.0 1.0 0.1
4 5 2 o
o 1 o o
1 o o o
1 5 2 o
2 1
o o 1 o
o 1 1 1
5 4 4 2
5 4 3 1
o o o 1
2 o o o
2 3 3 1
0/0 Root development
with GAa
10.0 1.0 0.1
10 0 40 30 60 10 60 20
o 0 o 50
50 50 o 20
o o o o
o o o o
40 0 0 40 50 0 50 10 0 40 0 0
000 o 50 0
20 40 30 o 20 30
without GAs
10.0 1.0 0.1
o 10 0 o 20 0
50 0 0 70 0 0
000 o 0 10 o 0 20
30 0 10
o 0 10 0 20 30 o 0
o 10 10 10 10 0 40 20
o o o o
o o o o
a) Grades: 5, very good; 4, good; 3, average; 2, poor; 1, very poor; 0, nil. b) GAa at 0.1 .uM level.
Z. Pjlanzenphysiol. Bd. 95. S. 51-56. 1979.
Cassava meristem and internode culture 55
the auxin requirement reported here may be due to differences of the endogenous auxin levels of the varieties used.
From the foregoing experiments it may be concluded that, for enhancing shoot differentiation, BA is superior to zeatin, kinetin and 2iP. The stimulatory effect of G A3 in shoot differentiation as reported earlier (KARTHA et al., 1974) has been confirmed.
Our observations indicated that not only the development but also the vigor of the shoots is critical for ensuring the growth of these plants to maturity. Thus the product of these two parameters referred to as «Regeneration index» would form a suitable basis for comparison (Table 2). It now became quite clear that GAs had a pronounced effect on the frequency of shoot differentiation when 2iP and kinetin were used.
The internodes of both «M 4» and «Kalikalan» produced callus, sometimes with roots. Callus production in explants from the internodes of the cultivar «M 4» was more extensive than in that of <Kalikalan>. Although some of the callus turned
Table 2: Effect of growth regulators on the regeneration index'f )
from cassava meristems. of plantlets regenerated
Hormone levels CuM) With GA3(0.1 ,uM) Without GAs
NAA 10.0 1.0 0.1 10.0 1.0 0.1
Zeatin 10.0 40 45 30 16 20 45
1.0 16 16 50 10 8 40 0.5 2 6 27 4 9 18 0.1 2 14 0 7 8
2iP 10.0 7 5 18 0 0 0
1.0 3 3 10 1 0 0 0.5 2 4 10 0 1 0 0.1 0 0 20 0 0 1
Kinetin 10.0 20 0 20 3 0 18 1.0 2 6 20 0 3 0 0.5 7 6 20 0 9 0 0.1 4 4 10 0 5 0
BA 10.0 6 50 30 2 50 20 1.0 36 40 50 35 40 30 0.5 50 30 50 16 40 30 0.1 20 20 40 0 16 10
") Regeneration index: number of plants regenerated X plant vigor grade.
Z. Pjlanzenphysiol. Bd. 95. S. 51-56. 1979.
56 N. G. NAIR, K. K. KARTHA, and O. L. GAMBORG
green upon transfer to GA-free medium, they did not form any shoots. Therefore, our results on plant regeneration from the internodal callus are not in agreement with those of TILQUIN (1978) who reported to have induced shoot regeneration from cassava callus derived from internode culture. Moreover, previous attempts to induce shoot differentiation from the callus derived from petioles, leaf laminae, and internodal segments of 10 Colombian, one Nigerian and one Indian cassava cultivars were also unsuccessful (KARTHA, unpublished).
Acknowledgements
A research training scholarship granted to one of the authors (NGN) by the International Development Research Centre, Ottawa, Canada is gratefully acknowledged. The views expressed in this publication are those of the authors and do not necessarily reflect the views of the International Development Research Centre. Our grateful thanks are also due to Dr. N. HRISHI, Director, Central Tuber Crops Research Institute, Trivandrum, India, for the supply of cassava material and also for his keen interest in this research project.
References
BOCK, K. R. and E. J. GUTHRIE: Transmission of African cassava mosaic by mechanical inoculation. Plant Dis. Reptr. 62, 580-581 (1978).
KARTHA, K. K., O. 1. GAMBORG, F. CONSTABEL, and J. P. SHYLUK: Regeneration of cassava plants from apical meristems. Plant Sci. Lett. 2,107-113 (1974).
KARTHA, K. K. and O. 1. GAMBORG: Elimination of cassava mosaic disease by meristem culture. Phytopathology 65, 826-828 (1975).
MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15, 473-497.
TILQUIN, J. P.: Regeneration and rapid multiplication of cassava by internode and callus culture. (Abstr.). International symposium on diseases of tropical food crops, Universite Catholique, Louvain-Ia-Neuve, Belgium 1978.
Dr. K. K. KARTHA and Dr. O. 1. GAMBORG, Prairie Regional Laboratory, National Research Council of Canada, Saskatoon, Saskatchewan, S7N OW9, Canada.
Z. P/lanzenphysiol. Bd. 95. S. 51-56. 1979.
