International Journal of Agricultural

Science and Research (IJASR)

ISSN(P): 2250-0057; ISSN(E): 2321-0087
Vol. 6, Issue 5, Oct 2016, 269-276
TJPRC Pvt. Ltd

EFFICIENT IN-VITRO REGENERATION OF PEARL MILLET

(PENNISETUM GLAUCUM (L.) FROM MATURE EMBRYO DERIVED CALLUS
KRISHNA KUMAR DWIVEDI1, A. RADHAKRISHNA2, P. KAUSHAL3 & R. KATIYAR4
1

Senior Scientist, Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi, (UP), India
2

3
4

Scientist, Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi, (UP), India

Principal Scientist, Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi, (UP), India

Senior Research Fellow, Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi, (UP), India

ABSTRACT
An efficient system was developed for in vitro plant regeneration via somatic embryogenesis using germinating
seeds of Pennisetum glaucum (L.) genotypes P31617, AVKB19 and D5/302. We tested different media supplements effect
on callus induction, somatic embryogenesis, shoot induction and root formation on different genotypes. The mature
embryo (seeds) of P. glaucum were cultured on Murashige and Skoog (MS) medium supplemented with a combination
of 3.5-4.5mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 0.5-1.0 mg/l kinetin for callus induction. Genotype P31617
genotypes. Hard and compact embryogenic calli were transferred on MS media supplemented with 0.5-2 mg/l 2, 4-D and
1-3 mg/l kinetin for somatic embryogenesis. The maximum rate of embryogenic callus was obtained from genotype
P31617 at 0.5 mg/l 2, 4-D and 2mg/l kinetin concentration in MS medium. Embryogenic calli were incubated at 25C
under an alternative photoperiod scheme (16h light/ 8h dark) on same media. The regenerated shoots were transferred
on to MS medium supplemented with 23 mg/l indole-3-acetic acid (IAA) and 13 mg/l kinetin for plant regeneration
and rooting. The maximum shoot induction frequency (70%) and root induction frequency (40%) observed in genotype

Original Article

showed highest callus induction frequency (80%) at 4.5mg/l 2.4-D with 0.5 kinetin in MS media as compared to other

P31617 at MS + 3 mg/l IAA + 2 mg/l kinetin. The rooted plantlets were hardened and transplanted to pots, where they
exhibited morphologically normal growth.
KEYWORDS: Pearl Millet, Somatic Embryogenesis, Callus Induction Frequency, Mature Embryo

Received: Sep 02, 2016; Accepted: Sep 26, 2016; Published: Sep 28, 2016; Paper Id.: IJASROCT201631

INTRODUCTION
Pearl millet [Pennisetum glaucum (L.) R. Br] is a drought tolerant cereal crop used as feed or forage crop
(Goldman et al., 2003). It is cultivated in semi-arid tropical region of Africa and Asia and covers about 40 million
ha of land in that region (Lamble et al., 2000). It can be grown especially under low soil fertility and harsh
climatic conditions where other cereals such as maize and wheat cannot be grown (Arockiasamy et al., 2006).
Pearl millet grains are rich in protein (12.3%), fats (5.3%), essential amino acid such as leucine (2-4%) and other
minerals (Bashy 1996). Because of high nutritive value, it is recommended for diabetic and cardiac patients
(Anonymous 1959). It is an important forage crops for ruminants. P. millet is regarded as a high quality feed for
grazing, green chop, hay, or silage. It is high in protein and energy and low in fiber and lignin concentration
(Smith et al., 1988). It can be used either under continuous or rotational grazing, however, the most efficient way
to use this valuable forage resource is to graze it rotationally (Newman et al., 2010). It is highly susceptible to

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Krishna Kumar Dwivedi, A. Radhakrishna, P. Kaushal & R. Katiyar

downy mildew disease which can cause up to 70% reduction in yield (Pinard et al., 1989). Insect pest also inflict severe
damage in field and during storage. Therefore, improvement in this crop for biotic and abiotic stress will have a great effect
on socio-economic status of people in the rural area where it is widely cultivated.
Efficient plant regeneration is a prerequisite for complete genetic transformation for the genetic improvement of
this crop plant. Several reports of in vitro differentiation from various pearl millet explants, such as Immature embryos
(Vasil and Vasil ,1981; Oldach et al.,2001), Immature inflorescence (Vasil and Vasil,1981; Botti and Vasil ,1983 ;
Pius et al.,1993 ; Mythili et al.,1997), Leaf bases (Haydu and Vasil ,1981; Rajasahkheran et al.,1987), Shoot apices
(Devi et al.,2000), Mature embryos (Jha et al.,2009; Rishi et al., 2012). However improvement in culture conditions to
simplify the producer for induction of embryogenic calli and to increase both the frequency of somatic embryogenesis and
the conversion of embryos to plants is desirable. Here we report a highly reproducible and efficient plant regeneration
protocol which is based on organogenesis and indirect somatic embryogenesis from mature embryo explants.

MATERIALS AND METHODS

Experimental Materials
Mature and healthy seeds of P. glaucum genotypes P31617, AVKB19 and D5/302, available at Central Research
Farm of Indian Grassland and Fodder Research Institute, Jhansi, India were used in the present study. We tested different
media supplements effect on callus induction, somatic embryogenesis, shoot induction and root formation on P. glaucum
genotypes.
Callus Induction
The mature seeds were manually dehusked, sterilized in 70% ethanol for 3min and in a 0.2% HgCl2 solution for
5-10min. They were then washed 3-5 times in sterilized water. Surface sterilized seeds were planted onto petri dishes
(90 X 15 mm) containing 25ml MS media (Murashige and Skoog 1962) supplemented with 500mg/l casein hydrolysate,
0.3g/l proline, 0.4g/l cystine, 30g/l sucrose, and supplemented with a combination of 3.5-4.5 mg/l (2, 4-D) and 0.5 mg/l
(kinetin or BAP) in different combinations for callus induction in darkness at 250C. For each treatment 20-25 seeds were
planted onto 3 petri dishes with a total of 3 replicates per treatment.
Callus Growth and Quality Assessment
Callus induction frequencies per seed were determined after 15-20 d of inoculation. Calli were then transferred to
new media, and this process was repeated at two week intervals for up to four times. Morphogenic changes, including
callus texture, were recorded at each sub culturing and correlated with media components. Callus color was recorded as ice
white and brown, yellow and light brown, white (creamy), milky white, or egg white. Callus texture was recorded as
vitreous, fragile, friable, compact, nodular, or hard.
Somatic Embryogenesis
After 4-6 weeks of culture initiation, hard nodular and light brownish calli induced on each of the induction media
was transferred to media with various concentration of BAP (2to 3 mg/l) or Kinetin (2to 3 mg/l) in combination with 2,4-D
(0.5mg/l) for embryogenesis. For shoot regeneration, embryogenic callus were incubated at 250C under an alternative
photoperiod scheme (16 h light/8h dark) on the same media. For each treatment 15-20 embryogenic calli were planted on
regeneration media in three conical flasks (100ml) as one replicate, with three replicates performed. Embryogenic callus
Impact Factor (JCC): 4.8136

NAAS Rating: 3.53

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271

induction frequencies and shoot regeneration frequencies were recorded.

Plant Regeneration, Rooting, Hardening and Acclimatization
After 3-4 weeks, the regenerated shoots were transformed on to MS medium supplemented with various
concentration of IAA (2 to 3mg/l) with varying concentration of Kinetin (1, 2, 3mg/l) for rooting under the same condition.
Following incubation for 2 weeks, the plantlets were removed from the culture flasks and washed to remove adhering agar
from the roots. The plantlets were then placed in test tubes containing sterile water for 2-3 d and subsequently transferred
into pots containing soil rite. Root induction frequencies and mean numbers of roots formed per shoot were recorded.
Data Analysis
The experiments were performed using various combinations of hormones, each treatment consisting of six
successfully-callused explants per petridish in three replications. Each experiment was repeated twice. The data were
analyzed statistically using SPSS ver.10 (SPSS, Chicago, IL).Significant differences among means were identified by two
way analysis of variance using the least significant difference procedure (P0.05).

RESULTS AND DISCUSSIONS

Callus Induction
After two week of culture seed were leading to multiplication of cell by producing a mass of callus.
Callus induction frequency was observed. The highest callus induction frequency (80%) was observed in P31617 genotype
at 4.5 ppm 2,4-D in combination of 0.5 ppm Kinetin in MS medium (Table 1). Mythili et al., 1997 reported the frequency
of embryogenic callus induction in 3 genotypes ranged from 78-87% from shoot tips. Whereas Jha et al., 2009 reported
20-85% in different genotypes with 2,4-D alone or combination with BAP in different explants. Arockiasamy et al., 2006
also reported that 2,4-D alone gave up to 75.0% callus induction frequency from mature embryos and 42.0% from leaf
explants. It indicated that endogenous Auxins levels were varying from genotype to genotype in P. glaucum which
strongly influence the callus initiation. Overall genotype P31617 showed better callus initiation response in comparison to
other two genotypes.
Somatic Embryogenesis
At initial stage watery and loose mass of calli were produced. Repeated sub-culturing of these callus in MS media
containing varying concentration 2, 4-D and kinetin or BAP initiated improved quality of callus growth, color and texture
(Figure 4A).The continuous sub culturing of callus led to the formation of hard and compact embryogenic callus
(Figure 4B).
Shoot Regeneration
Hard and compact embryogenic calli were transferred on MS media supplemented with 0.5 mg/l 2, 4-D and 1-3
mg/l kinetin for shoot induction (Figure 4 C). Maximum frequency of shoot regeneration (70%) was recorded in 0.5ppm
2,4-D + 2ppm Kinetin in MS media, from callus tissues derived from P31617 (Table 2). Arockiasamy et al., 2006 also
reported the shoot induction frequency (70%) with kinetin and BAP. Jha et al., 2009 reported 23% with alone BAP from
mature embryos. A significant genotypic difference was also observed in regeneration capacity in sorghum.

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Krishna Kumar Dwivedi, A. Radhakrishna, P. Kaushal & R. Katiyar

Rhizogenesis and Hardening

The regenerated shoots were transferred on to MS medium supplemented with 23 mg/l IAA and 13 mg/l kinetin
for plant regeneration and rooting (Figure 4 D). The maximum root induction frequency (40%) was observed in genotype
P31617 at MS + 3ppm IAA + 2ppm Kinetin media combination (Table 3). Well rooted plants were hardened, transferred to
pots, they appeared normal when compared to non-regenerated mother plants (Figure 4 E). Arockiasamy et al., 2006 found
IBA was best for root induction, however the frequency of root formation varied with different concentrations of various
auxins.
In conclusion, we have optimized the efficiency and reproducibility of in vitro procedures for high frequency
plant regeneration of Pearl millet from seed explants through callus derived somatic embryogenesis. This report also
suggests the possibility of improving regeneration procedures for specific genotypes. Our regeneration system provides a
foundation for the genetic transformation of Pearl millet, thus enabling genetic engineering improvements of this important
forage crop.

CONCLUSIONS
In the present study, we have successfully developed the in vitro plant regeneration system via somatic
embryogenesis using germinating seeds. We tested different media combination for callus induction and regeneration on
different genotypes. A highly efficient media combination has been optimized to achieve as high as 80% for callus
induction, 70% shoot induction and 40% root induction frequency from mature seeds of P. glaucum. This information may
be important in view of generating transgenics in this important crop.

ACKNOWLEDGMENTS
This work was supported by Department of Biotechnology (DBT) in from of Rapid Grant for Young Investigators
(RGYI 2012) (BT/PR6008/GBD/27/384/2012).
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Bashy,A.Y., (1996) Nutritional and chemical evaluation of pearl millet grains (Pennisetum typhoides (Burm.f) Stapf and Hubb,
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Botti, C., Vasil, I.K.,(1983) Plant regeneration by somatic embryogenesis from parts of cultured mature embryos of
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Impact Factor (JCC): 4.8136

NAAS Rating: 3.53

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Haydu,Z., Vasil,I.K.,(1981) Somatic embryogenesis and plant regeneration from leaf tissues and anthers of Pennisetum
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Jha,P., Yadav,C.B., Anjaiah,V., Bhat,V., (2009) In vitro plant regeneration through somatic embryogenesis and direct shoot
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Derived Callus. IJASR 2:37-40
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20. Vasil, V., Vasil,I.K.,(1981) Somatic embryogenesis and plant regeneration from tissue cultures of Pennisetum americanum and
P.americanum P. purpureum hybrid. Am J Bot 68:864872

APPENDICES

Figure 1: Callus Induction Frequency (%) Se in Mature Embryo on Ms Media

Containing Combination of A, 3.5( Mg/L) 2-4d +0.5(Mg/L) Bap; B, 4.5 (Mg/L) 2-4d +0.5(Mg/L)
Bap ;C, 3.5 (Mg/L) 2-4d +0.5(Mg/L) K ;D, 4.5 (Mg/L) 2-4d +0.5(Mg/L) K
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Krishna Kumar Dwivedi, A. Radhakrishna, P. Kaushal & R. Katiyar

Figure 2: Shoot Induction Response of Calli on MS Medium with Combination of BAP and
Kinetin A, 2(Mg/L) BAP; B, 3(Mg/L) BAP; C, 3(Mg/L) K; D, 0.5(Mg/L) 2-4D+ 2 (Mg/L) K

Figure 3: Root Induction Frequency (%) SE In MS Media Containing Variable Concentration

Of Auxin And Cytokine A; 2(Mg/L) IAA+2 (Mg/L) K; B, 2(Mg/L) IAA+3 (Mg/L) K; C, 3(Mg/L)
IAA+2(Mg/L) K; D, 3(Mg/L) IAA+1 (Mg/L) K

Impact Factor (JCC): 4.8136

NAAS Rating: 3.53

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275

Figure 4: In Vitro Plant Regeneration in P. Glaucum Genotype P31617. A: Callus Induction; B: Somatic
Embryogenesis; C: Shoot Induction; D: Root Induction and Shoot Elongation; E: Plant Transferred in Pot
Table 1: Effect of 2, 4-D, BAP and Kinetin on Callus Induction (%) from Mature Embryo on MS Medium
Genotype
P31617

AVKB19

D5/302

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Concentration of
Hormone(mg/l)
3.5D+0.5K
4.5D+0.5K
3.5D+0.5B
4.5D+0.5B

Callus Induction
Frequency (%) SD
511.7
79.40.5
581.9
43.31.4

3.5D+0.5K
4.5D+0.5K
3.5D+0.5B
4.5D+0.5B

48.52.6
68.91.0
52.01.8
37.91.8

3.5D+0.5K
4.5D+0.5K
3.5D+0.5B
4.5D+0.5B

41.11.0
56.62.6
55.71.0
32.61.2
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Table 2: Morphogenetic Responses of the Calli of Mature Embryo on

MS Media Supplemented with Different Concentration of BAP and Kinetin
Genotype
P31617

AVKB19

D5/302

Concentration of
Hormone (Mg/L)
2BAP
0.5D+2K
3K
3BAP

Shoot Induction
Frequency (%) SD
53.02.6
70.91.6
42.22.8
51.11.9

2BAP
0.5D+2K
3K
3BAP

50.00.0
59.41.9
32.21.9
41.21.0

2BAP
0.5D+2K
3K
3BAP

44.44.8
52.84.8
30.00.0
59.41.0

Table 3: Effect of Auxin and Cytokine on Root Induction in Regenerated

Shoots of Pennisetum Glaucum on MS Medium
Genotype
P31617

AVKB19

D5/302

Impact Factor (JCC): 4.8136

Concentration of
Hormone(Mg/L)
2IAA+2K
3IAA+2K
3IAA+1K
2IAA+3K

Root Induction
Frequency(%) SD
21.72.9
41.13.1
34.72.4
26.22.1

2IAA+2K
3IAA+2K
3IAA+1K
2IAA+3K

33.30.0
37.50.0
36.12.4
28.32.9

2IAA+2K
3IAA+2K
3IAA+1K
2IAA+3K

36.12.4
23.11.6
30.00.0
19.82.8

NAAS Rating: 3.53