PlantCeU Reports

Plant Cell Reports (1988) 7:557-559

© Springer-Verlag1988

Callus induction and high frequency plant regeneration in Italian millet (Setaria italica) A.M. Rao 1, p.B. Kavi Kishor 1, L. Ananda Reddy 2, and K. Vaidyanath 2 1 Department of Botany, Kakatiya University, Warangal 506009, India 2 Department of Genetics, Osmania University, Hyderabad 500007, India Received May 25, 1988/Revised version received September 8, 1988 - Communicated by I. K. Vasil

Abstract Callus was induced from mature seeds of two cultivars of Setaria italica (L.) on Murashige and Skoogls medium (1962) supplemented with 2mg/l 2,4-D and 0.5 mg/l KN. Regenerating ability of the callus was better in the cultivar 315 compared to 212. Organogenesis was influenced not only by cytokinin, but also by the sucrose concentration in the medium. High frequency (80%) plant regeneration was achieved and quantified on the basis of callus fresh weight. The ability of the callus (cultivar 212 ) to regenerate whole plants was retained until the 5th passage, but during the 6th passage it declined considerably. Abbreviations 2,4-D=2,4-dichlorophenoxyacetic Kinetin, BAP = 6-benzylaminopurine, and Skoog basal medium.

acid, KN= MS=Murashige

Italian millet (Setaria italica (L.) P.Beauv.) is an important arid crop grown mainly in India, Asia and USA. The grain is used as food after husking and is eaten in the form of cakes or porridge. It is valued as a flour for making pastry. When boiled with milk, it forms a light and pleasant meal for invalids. The straw is thin and leafy and is good as fodder (Anonymous,1972). Callus formation and regeneration of plants have been achieved in a number of cereals (Vasil and Vasil,1986). To date no report has yet d o cumented plant regeneration f r o m callus cultures of Italian millet. The present paper describes induction and maintenance of callus from mature seeds of Italian millet and plant regeneration. Materials and Methods Mature seeds of the cultivars ISE 212 and ISE 315 were surface sterilized with 0. 1% mercuric chloride for 12-15 rain and washed thoroughly with sterile distilled water. Two to three seeds were inoculated into glass tubes (60ml capacity) containing 15 ml of MS agar (0.8%) medium (Murashige and Skoog, 1962 ) supplemented with 2 mg/l 2,4-D, 0.5 mg/l KN plus 2% sucrose (hitherto referred to as IM). For callus initiation and maintenance, the cultures were incubated at 25-+2% under continuous, cool white

Offprint requests to: A.M. Rao

fluorescent light (1000 lux). Callus was obtained within two to three weeks from 80 to 95% of the seeds. Subcultures were done every 20 days on to the same medium (IM). Different concentrations of 2,4-D, BAP, KN and sucrose were tested to obtain m a x i m u m frequency of plantlets. For regeneration, cultures were incubated at 2 5 + 2 % under continuous, white fluorescent light (2000 lux). Experiments were repeated at least once. Results and Discussion Callus tissues of cultivar ISE 212 and subsequent regeneration of plantlets are shown in Figs. I-3. Callus production was generally low in both the cultivars used regardless of the concentration of 2,4-D or KN. On an average 65+-14 mg of fresh callus was obtained from each seed during the first passage (20 days) on MS medium. During the initial passage, callus looked watery and friable, but the callus became compact from the second passage onwards. Embryogenie callus, however, was never observed. After three subcultures of 20 days each, callus of both cultivars was transferred to MS basal media containing different concentrations of 2,4-D (Table I). Optimum frequency of plantlet regeneration in both the cultivars was noticed on medium containing 0.I mg/l 2,4-D plus 2 mg/l KN, compared to low (0.05 mg/l) and high (0.2 mg/l) 2,4-D media. However, cultivar ISE 315 showed better response (75%) compared to cultivar ISE 212 (50%). Regeneration of plantlets from callus cultures of ISE 212 is shown in Fig.2. The effect of KN, B A P and sucrose concentrations on callus regenerating ability (ISE 212) is represented in Table 2. Enhanced KN concentration in the medium increased the frequency of plantlet regeneration, the optimum concentration being 2 mg/l. The number of plantlets formed per 200 mg of fresh callus was also highest at 2 mg/l KN when compared to any other medium. While lower concentrations of BAP (0.5 and 1 rag/l) failed to induce any plantlets, higher concentrations (2 and 4 rag/l) produced only very small plantlets (Table 2).

558 Table I. Effect of 2,4-D on regenerating ability of S.italica callus cultures a

% frequency of pla~tlet production

Hormones mg/l

0.05 2,4-D + 2 K N 0.I 2,4-D + 2 K N 0.2 2,4-D + 2 K N

b%

ISE 212

ISE 315

40 50 35

50 75 40

Relative advantage of cultivar ISE 315 +10 c +25 c +05 c

were scored at the end of 25 days using 60 day old callus from 20 replicate cultures frequency of response = No.of calli with plantlets x I00

D

a

t

a

Total No. of calli inoculated CSignificant p = 0.I0 Table 2. Effect of KN,

BAP

and sucrose on regenerative capacity of S.italica cv.ISE 212 callus a

Hormones mg/l

0.5 I 2 4 0.5 i 2 4 2 2 2 2

K N + 0.I K N + 0.I KN + 0.I K N + 0.I B A P +0.I B A P + 0.i B A P + 0.I B A P + 0.I K N + 0.I K N + 0.I K N + 0.I K N + 0.1

2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D 2,4-D

Sucrose %

(Control)

(Control)

(Control)

2 2 2 2 2 2 2 2 0.5 1 2 4

% frequency of plantlet production

Mean (+SE) no.of plantlets formed per 200 m g callus

30 50 80 70 Nil Nil 50 30 Nil 20 80 80

7.1 8.3 27.0 12.5

16.8 12.1 3.8 26.7 6.9

(+0.5)~ (+0.7)~ (+0.5) c (+0.5) c (+0.4) c (+0.5) c (+0.2) c (-+0.5)~ (-+0.3)c

~Data

were scored at the end of 25 days using 90 day old callus and I0 replicates Not significantly different CSignificant p = 0.01

Fig.l Unorganised mass of callus induced from mature seeds of Italian miler CV.ISE 212.

Fig.2 Plant regeneration from callus cultures of ISE 212.

Fig.3 Well developed plantlets of ISE 212 before transfer to soil,

559 Sucrose at 0.5% did not evoke any organogenetic response. Plantlet formation was low (20%) at 1% sucrose, but 2% gave the best (80%) response. Further increase in sucrose concentration (4%) did not diminish the frequency of plantlet regeneration, but the number of plantlets produced per 200 mg of callus was reduced (Table 2). Regenerated plantlets of ISE 212 were transferred to MS basal s a l t s containing 2% sucrose, 1.5 mg/l KN and 1 mg/l naphthalene acetic acid for subsequent development of shoot and root (Fig.3) before transfer to soil. The ability of the callus to regenerate whole plants was retained until the 5th passage (80%), but during the 6th passage it declined considerably (40%). The results showed that KN was more effective for organogenesis compared to BAP. Such a finding was also observed in callus cultures of rice (Kavi Kishor, unpublished). Two percent sucrose seemed to be optimum for regeneration in Italian millet, as found in other cereals such as rice (Kavi Kishor,1987). Loss of totipotency after a few subcultures is common in many plants (Barba and Nickell, 1969; Lustinec and Horak,1970; Kavi Kishor and Reddy,1987). As postulated by Gautheret (1966), this could be because of the disappearance of a specific factor contained in the primary explant or due to polyploidisation (Sheridan,1975). A total of 200 to 240 plantlets were obtained from a single seed in a period of I00 days. The regenerated plants have been successfully transferred to pots and are now being assessed for somaclonal variation.

Acknowledgements A M R is thankful to the University Grants Commission, New Delhi, India for financial assistance. We thank the Head, Department of Botany, Kakatiya University, Warangal for providing the facilities. References Anonymous (1972) Wealth of India: R a w Materials, CSIR Publication and Information Directorate 9:304-312. Barba R, Nickell LG, (1969) Planta 89:299-302. Gautheret R J, (1966) In: Beerman W(ed) Cell Differentiation and Morphogenesis, North Holland, Amsterdam, pp 55-95. Kavi Kishor PB, (1987) Plant Sci 48:189-194. Kavi Kishor PB, Reddy GM, (1987) Ind J Plant Physiol 30:66-70. Lustinec J, Horak J, (1970) Experientia 26:919-920. Murashige T, Skoog F, (1962 ) Phy siol Plant 15:473-497. Sheridan WF, (1975) In: Ledoux L(ed) Genetic Manipulations with Plant Material, Plenum Press, New York, pp 263-295. Vasil IK, Vasil V, (1986) In: vasil IK (ed) Cell culture and Somatic Cell Genetics of Plants, Volume 3, Plant Regeneration and Genetic Variability, Academic Press, Orlando, pp 121-150.

Callus induction and high frequency plant regeneration in Italian millet (Setaria italica).

Callus was induced from mature seeds of two cultivars of Setaria italica (L.) on Murashige and Skoog's medium (1962) supplemented with 2mg/l 2,4-D and...
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