Industrial Crops and Products 67 (2015) 324–329

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Industrial Crops and Products

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Root explant produces multiple shoot from pericycle in Psoralea

corylifolia – a leprosy destroyer medicinal plant
Jigar N. Jani, Suman Kumar Jha ∗ , Durga Singh Nagar
Department of Forest Biology and Tree Improvement, College of Forestry, Navsari Agricultural University, Navsari, Gujarat 396 450, India

a r t i c l e i n f o a b s t r a c t

Article history: Babchi (Psoralea corylifolia L.) is a rare medicinal plant. It has been considered to be efficacious in leprosy
Received 28 October 2014 and hence given the name ‘Kushtanashini’ (leprosy destroyer) in Ayurveda. The high death rate in wild
Received in revised form 31 January 2015 along with aimless and unlawful gathering and destruction of habitat put the plant in the category of
Accepted 2 February 2015
endangered list. In this study a highly reproducible in vitro regeneration system in P. corylifolia using root
segments was developed. The effect of different PGRs (Kin BA, and NAA) on the percentage of adventitious
Keywords:
shoot induction and the number of shoot buds per root explant was assessed. The maximum response
Psoralea corylifolia
(65.57%) was observed in the media supplemented with 2.22 ␮M BAP and 6.98 ␮M Kin. The treatment
Direct regeneration
Root explant
had in average 39.67 number of shoot buds and 3.37 elongated shoots. Elongation response was best
Phloroglucinol (92.0%) in MS + 2.22 ␮M BAP + 6.98 ␮M Kin media fortified with 2.89 ␮M GA3 and 39.7 ␮M PG, producing
Pericycle a mean of 9.3 elongated shoots with an average of 4.6 cm length. An anatomical study showed that the
origin of shoot buds were directly from pericycle of root. Elongated shoots were carefully excised and
rooted on basal MS + 4.92 ␮M IBA + 158.6 ␮M PG, with 92 per cent rooting. Rooted explants survived in
soil: vermicompost: vermiculite (1:1:1) with 100% success.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction used in Ayurvedic and Chinese medicine system as a cardiac tonic,

vasodilator, pigmentor, antitumor, antibacterial, cytotoxic, antihel-
To battle diseases, medicinal plants have been an integral part menthic, laxative, aphrodisiac, diuretic and diaphoretic in febrile
of human society from the beginning of civilization. A few hundred conditions. The seeds of the plant are used in indigenous system for
genera of plants are utilized therapeutically as a household substi- the treatment of leucoderma, leprosy, psoriasis, hair loss, eczema,
tute in the indigenous system of prescription in different nations, bilious disorders, scorpion-sting and snake bite (Chopra et al., 2013;
and hence, present day solutions have not possessed the capacity Gidwani et al., 2010, 2011; Kapoor, 1989; Khare, 2004; Khushboo
to replace the greater part of them. In the last few decades, the et al., 2009; Krishnamurthi, 1969; Panda, 1999; Purkayastha and
field of herbal medicine has gotten popularized in both developed Dahiya, 2012). A wide range of chemical compounds, including
and developing countries. This is because the herbal medicines are psoralen, isopsoralen, bakuchiol, psoralidin, bakuchalcone, bava-
cheap, and have a natural origin with higher safety margins (Gupta, chinin, flavones, volatile oils, lipids etc. are found in different parts
2012). The World Health Organization reported that 80% of the of the plant (Boardley et al., 1986; Chopra et al., 2013). Psoralene
world populations depends mainly on traditional medicine, includ- and isopsoralene used widely as anticancinogen (Abhyankar et al.,
ing the utilization of plant concentrates or their active constituents 2005; Oliveira et al., 2006; Wang et al., 2011). Bakuchiol are used
(Duraipandiyan et al., 2006). in a variety of diseases (Haraguchi et al., 2000; Katsura et al., 2001;
Babchi (Psoralea corylifolia L.) is a rare medicinal plant, belong- Mao et al., 2014; Miao et al., 2013; Villegas et al., 2015). Recently
ing to family Fabaceae and native to South Eastern Asia and largely this compound has been reported to have anti ageing property
cultivated in China. It is distributed in tropical and subtropical (Chaudhuri and Bojanowski, 2014).
regions of the world. The plant has been considered to be effica- The high death rate in wild along with aimless and unlawful
cious in leprosy and hence given the name ‘Kushtanashini’ (leprosy gathering and destruction of habitat put the plant in the category
destroyer) in Ayurveda (Khushboo et al., 2009). It has been widely of endangered list (Baskaran and Jayabalan, 2007, 2008). There is
need for alternate propagation technique like in vitro regeneration
to save this important plant species. In vitro shoot culture could also
provide an alternative to field plant harvesting for the production
∗ Corresponding author. Tel.: +91 2637 282143; fax: +91 2637 282145.
of therapeutically valuable compounds (Jain et al., 2011; Sangwan
E-mail addresses: [email protected], [email protected] (S.K. Jha).

http://dx.doi.org/10.1016/j.indcrop.2015.02.001
0926-6690/© 2015 Elsevier B.V. All rights reserved.
 J.N. Jani et al. / Industrial Crops and Products 67 (2015) 324–329 325

et al., 2007). Although, many reports on in vitro regeneration from 2.3. Rooting and acclimatization of plantlets
various explants viz Shoot tips (Anis and Faisal, 2005; Baskaran and
Jayabalan, 2008), Nodal explants (Anis and Faisal, 2005; Baskaran Shoots 1.5–2.0 cm in length were excised and transferred to
and Jayabalan, 2007, 2008; Jeyakumar and Jayabalan, 2000), cotyle- half-strength MS medium supplemented with 4.92 ␮M IBA and
donary node (Jeyakumar and Jayabalan, 2002), axillary shoot (Faisal 158.6 ␮M PG. The rooted plants were removed from the culture
and Anis, 2006), apical meristem (Pandey et al., 2013) and cal- tubes, washed free of agar with sterile distilled water and trans-
lus through regeneration (Baskaran and Jayabalan, 2009; Saxena ferred to plastic pots with sterile potting mixture for hardening.
et al., 1997; Shinde et al., 2010) is available in this species, only a The plantlets were maintained at 70% relative humidity by ini-
few authors reported on direct shoot regeneration (Baskaran and tially covering with transparent polyrthene. The plants were kept
Jayabalan, 2010). So far, in our knowledge, nil account on direct in 28 ◦ C under a 12-h photoperiod for acclimatization. The plants
adventitious shoot regeneration from root explants is available in were fertilized with 1/8th MS macro nutrients twice during the
this species. Besides, in the medicinal plants, many questions like course of acclimatization at an interval of 4–5 week. Established
understanding the pathways of metabolite and metabolic engineer- plants were placed in 20 cm diameter pots with soil: vermicom-
ing for overexpression or knockdown of gene needs research. Most post: vermiculite mixture (1:1:1). These hardened plants were
of these can be answered using the efficient genetic transformation field-transferred.
technique (Li et al., 2008). Establishing direct plant regeneration
pathway is a key step in the application of genetic transforma-
2.4. Microscopic observation
tion technique for metabolic pathway manipulation in medicinal
plants (Verma and Mathur, 2011). Moreover, it is a faster way of
For microscopic observations, the samples of P. corylifolia roots
multiplying clones precluding somaclonal variation (Krishna et al.,
directly origination shoot bud sectioned and stained with safranin,
2010)
and used for microscope examination (Olympus, Tokyo, Japan)
Therefore, we describe an approach for high frequency shoot
regeneration from root explants and the ontogeny of direct shoot
formation from the root. Histological observation will strengthen 3. Results
our understanding of the regeneration process, which may further
help in designing the genetic transformation process. The morphogenetic response of root explants to various growth
regulators (Kin BAP and NAA) was evaluated. Root explants cul-
tured on growth regulator-free basal MS medium showed no sign
2. Material and methods
of bud break even after 30 d. The addition of a cytokinin was essen-
tial to induce bud break and multiple shoot formation from the
2.1. Explant preparation and culture establishment
explants. All levels of Kin BA, NAA alone or in combination formed
adventitious shoot bud on root explants (Table 1). After 1 week
Fresh and mature seeds of P. corylifolia were collected from
culture, organogenic response observed in the form of small per-
the medicinal plant garden at ASPEE College of Horticulture and
turbation of green shoot bud. Distinguishable shoot buds cluster
Forestry, Navsari Agricultural University, India. The freshly col-
appeared on all responsive root by the end of the third week. Vari-
lected seeds were washed under running tap water to remove
ous stages of shoot bud development from root explants is shown in
superficial contamination. The seeds were further washed with
Fig. 1a–c. Overall, Kin was more effective in combination with BAP
Tween-20 (6%, v/v) for 5 min followed by thorough washing under
for induction of multiple shoot bud and shoot development. The
running tap water for 15 min. Seeds were furher surface steril-
maximum response (65.57%) observed in the media supplemented
ized with mercuric chloride (0.1% w/v) for 7–8 min and rinsed 3–4
with 2.22 ␮M BAP and 6.98 ␮M Kin with an average of 39.67 shoot
times with sterile double distilled water. Then after, the seeds were
bud and 3.37 elongated shoots (Table 1). Addition of NAA reduced
dipped in 75% alcohol and flame heated to burn the extraneous
shoot bud induced callus formation.
seed coat adherents. The surface-disinfected seeds were germi-
For the development of economic viable protocol, number of
nated aseptically in culture bottle (200 ml, Borosil, India) containing
elongated shoots produced in each subculture are more important
growth regulator-free Murashige and Skoog (MS) (Murashige and
than the number of shoot bud per explants, since elongated shoots
Skoog, 1962) medium with 3% (w/v) sucrose (Hi-media, India) and
are only used for rooting and further regeneration cycle. Shoot buds
0.7% agar (bacteriological grade, Hi-media, India). Ninety percent
failed to develop in elongated shoots in abundant quantities in
of seed germinated within 15 days culture. Isolated root from
induction media. Hence, the effect of GA3 and Phloroglucinol (PG)
15 days old ascetically grown seedlings were cultured on full
investigated for shoot bud multiplication and elongation. Respon-
strength MS media supplemented with 4.44 ␮M and 6.66 ␮M BAP
sive root segments with buds isolated carefully and placed on basal
(6-Benzylaminopurine), 4.65 ␮M and 6.98 ␮M Kin (Kinetin) alone
MS + 2.22 ␮M BAP + 6.98 ␮M Kin supplemented with various con-
or 6.98 ␮M Kin in combination with 2.22 ␮M and 4.44 ␮M BAP and
centrations of GA3 and PG alone or in combination. The number of
2.69 ␮M NAA (Napthalene Acetic Acid). Data on number of explants
elongated shoots per responsive explant (multiplication rate) was
responding for shoot bud induction, average number of shoot bud
recorded four weeks after culture. The result showed that elonga-
per explants and number of elongated shoot were recorded after
tion response was best (92.0%) in MS + 2. 22 ␮M BAP + 6.98 ␮M Kin
four weeks of culture.
supplemented with 2.89 ␮M GA3 and 39.7 ␮M PG, which produced
an average of 9.3 elongated shoots with an average of 4.6 cm length
2.2. Shoot bud elongation (Table 2; Fig. 1d and e). Anatomy of root revealed that shoot were
originated from pericycle. Abundant accumulation of starch grain
Explants were cultured in shoot bud induction medium observed during bud formation (Fig. 1h, black spots evident in the
(MS + 2.22 ␮M BAP + 6.98 ␮M Kin) supplemented with 0.29 ␮M, figure)
1.45, 2.89 and 4.34 ␮M Gibberellic acid (GA3 ), 15.9 ␮M, 39.7 ␮M Elongated shoots were carefully excised and rooted on
and 79.4 ␮M Phloroglucinol (PG) or PG in combination with previously established rooting media (basal MS + 4.92 ␮M
2.89 ␮M GA3 . Observations on the number of explants responding IBA + 158.6 ␮M PG) for P. corylifolia in our laboratory, with 92
for shoot bud elongation, average number of elongated shoots and per cent rooting. All the rooted plantlets survived successfully in
average shoot length were recorded after four weeks of culture. the soil: sand: vermiculite (1:1:1) (Fig. 1f and g).
326 J.N. Jani et al. / Industrial Crops and Products 67 (2015) 324–329

Table 1
Effect of different growth regulators on percent response for direct shoot bud induction, number of shoot buds per explants and number of elongated shoot per culture in P.
corylifolia root culturea .

BAP Kin (␮M) NAA(␮M) Percent response Number of shoot Number of

(␮M) bud/explant elongated shoot

– – – 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00

4.44 – – 28.90 ± 1.10 14.67 ± 0.67 1.53 ± 0.09
6.66 – – 17.80 ± 1.10 19.67 ± 0.88 1.63 ± 0.09
– 4.65 – 25.57 ± 1.13 16.67 ± 0.88 1.47 ± 0.15
– 6.98 – 43.33 ± 3.33 35.67 ± 0.33 2.10 ± 0.40
2.22 6.98 – 65.57 ± 1.13 39.67 ± 1.45 3.37 ± 0.02
4.44 6.98 – 21.10 ± 2.20 21.33 ± 0.67 1.53 ± 0.13
2.22 6.98 2.69 24.43 ± 1.13 15.33 ± 0.67 1.73 ± 0.03
4.44 6.98 2.69 21.10 ± 1.10 14.33 ± 0.33 1.60 ± 0.15
CD0.05 – – 4.83 2.28 0.49
a
Data were recorded after four weeks of culture.

Fig. 1. In vitro regeneration of Psoralea corylifolia from root explants. (a–c) Initiation of shoot bud on root explants, (d–e) multiplication of shoot bud (f) rooting of microshoots
(e) hardening (f) anatomy of root producing direct shoot (black spot shows starch deposition).
 J.N. Jani et al. / Industrial Crops and Products 67 (2015) 324–329 327

Table 2
Effect of addition of GA3 and PG in MS + 2.22 ␮M BAP + 6.98 ␮M Kin media on per cent rensonse for shoot bud elongation, number of elongated shoot and average length of
elongated shoot per culture in P. corylifolia root culturea .

GA3 PG(␮M) Percent response Number of Average shoot

(␮M) elongated shoot length (cm)

0.29 – 43.3 ± 1.7 3.3 ± 0.3 1.7 ± 0.1

1.45 – 62.0 ± 1.2 4.3 ± 0.3 3.4 ± 0.2
2.89 – 58.0 ± 0.6 3.8 ± 0.4 2.8 ± 0.1
4.34 – 47.0 ± 1.0 3.3 ± 0.3 1.5 ± 0.2
– 15.9 62.3 ± 0.3 6.3 ± 0.3 2.4 ± 0.1
– 39.7 72.0 ± 1.2 6.8 ± 0.6 3.7 ± 0.1
– 79.4 67.7 ± 1.8 4.7 ± 0.3 3.1 ± 0.2
2.89 15.9 86.3 ± 0.9 5.7 ± 0.3 3.7 ± 0.3
2.89 39.7 92.0 ± 1.2 9.3 ± 0.3 4.6 ± 0.1
2.89 79.4 77.3 ± 0.7 6.3 ± 0.3 4.2 ± 0.1
CD0.05 3.32 1.12 0.44
a
Data were recorded after four weeks of culture.

4. Discussion phloridzin, is known for its growth regulating property (James,

1979; James et al., 1980). It acts as an auxin synergist during the
Direct multiple shoot induction is the useful means of produc- auxin-sensitive phase of organogenesis (James and Thurbon, 1981).
tion of plantlet from young or mature trees with a lower risk of It has the ability to enhance survival and shoot formation from small
genetic instability than by the other regeneration routes, hence tissue explants like meristems, and or shoot tips in vitro (Kumar
direct regeneration without intervening callus phase, is a more reli- et al., 2005). The effectiveness of certain phenolic substances in
able method for clonal propagation (Rao et al., 1996; Thorpe, 1994). enhancing shoot formation is well established in plant tissue cul-
Adventitious shoot regeneration is also most preferred regener- tures (Jones, 1976). Better shoot bud elongation has been reported
ation pathway if Agrobacterium-mediated gene transfer is to be when PG applied in combination with GA3 (Demiralay et al., 1997)
achieved (Kantia and Kothari, 2002). The buds formed on the roots of P. corylifolia via direct organo-
The results presented above clearly demonstrates that the high genesis originated from the pericycle cells; the similar result was
frequency shoot bud and plantlet regeneration can be achieved obtained in Passiflora edulis (Rocha et al., 2012). The pericycle has
from root explants derived from axenic grown seedlings in Pso- been proposed to be an extended meristem in the plant body (De
ralea corylifolia. Root explants exhibited shoot bud induction within Smet et al., 2006; Evert, 2006). A number of studies have reported
a week of culture. Root tissue has been proven to be highly regen- the involvement of the pericycle in the regeneration of shoots from
erative explant and is relatively easy to maintain and manipulate root explants (Arora et al., 2011; Lombardi et al., 2007; Vila et al.,
in vitro (Franklin et al., 2004; Vinocur et al., 2000). Reports are 2005; Vinocur et al., 2000), which is consistent with the current
also available in different species where root explants success- knowledge on the molecular mechanisms involved in the induc-
fully regenerated plant (Espinoza and Dodds, 1985; Hoshino and tion of shoot meristems in roots. Atta et al. (2009) demonstrated
Mii, 1998; Lazzeri and Dunwell, 1984; Shahidul Haque et al., 1997; the ability of pericycle cells to rapidly re-enter the cell cycle on
Sharma and Thorpe, 1989; Yoshimatsu and Shimomura, 1994). induction with cytokinin and maintain diploidy after several cycles
Explants could not show any sign of organogenesis in the of division, thus maintaining the genetic fidelity of the regenerated
absence of cytokinin, however, all the concentrations of cytokinin plants. Pericycle cells are lateral root initiation sites (Casimiro et al.,
evoked response. Cytokinin has already shown significant effects 2003; De Smet et al., 2006); however, under proper conditions, they
on shoot regeneration in P. corylifolia from different explants (Faisal may also be involved in the formation of shoot meristems. Regen-
and Anis, 2006; Jeyakumar and Jayabalan, 2002; Shinde et al., 2009). eration systems, such as those in the present study, in which root
Kin promote better direct shoot bud induction as compare to BAP. explants are cultured on media supplemented with only cytokinin,
Kin was found to better for direct shoot regeneration from root molecular studies have shown that the competence to form buds is
explants in Brassica napus (Sharma and Thorpe, 1989) however, acquired at sites where the spontaneous formation of lateral roots
in Diospyros kaki, zeatin in combination with IAA was the most occurs (Atta et al., 2009; Motte et al., 2011). Accumulation of starch
effective in stimulating the production of adventitious shoots from observed in the cell directly involved in bud formation. Accumula-
excised root culture (Tetsumura and Yukinaga, 1996). Multiple tion of starch in bud forming tissue and its utilization during bud
shoot response was better with Kin as compare to BAP in cotton break has been initially observed by Thorpe and Murashige (1968)
(Khan and Obembe, 2011). The synergistic positive effect of BAP in tobacco, thereafter many researchers reported this phenomena
and Kin was quite evident in the present study. The combination of in other species (Sharma et al., 1993). A degradation product of the
two cytokinins has also been found to be more effective than alone starch, along with free sugar in the medium are utilized as an energy
(Chalupa, 1987; Nielsen et al., 1995). Shoot bud induction response source for the high energy requiring process (Thorpe, 1980; Brown
(65.57%) and number of shoot buds (39.67) was highest in basal and Thorpe, 1986).
MS +2.22 ␮M BAP+ 6.98 ␮M Kin albeit a less number of elongated For rooting, when microshoots were placed in MS medium
shoots produced per culture. fortified with 4.92 ␮M IBA and 158.6 ␮M PG, adventitious roots
Induced shoot bud failed to develop in a shoot in abundant originated in large number from the cut end of the microshoot. All
quantities in induction media. Hence, the effect of GA3 and PG the rooted microshoots successfully hardened in soil: vermicom-
investigated for the production of the multiple elongated shoot post: vermiculite (1:1:1).
from each explants. Elongation response was best (92.0%) in
MS + 2.22 ␮M BAP + 6.98 ␮M Kin media added with 2.89 ␮M GA3
5. Conclusions
and 39.7 ␮M PG, producing mean 9.3 elongated shoots with an
average of 4.6 cm length. Supplementation of PG in the medium
In conclusion, to our knowledge the present study is the first
showed an enhancement in shoot bud and the quality of the shoot.
report for in vitro shoot regeneration of Psoralea corylifolia from
PG (1,3,5 trihydroxybenzene), one of the degradation products of
root explants. The procedure described here provides a rapid and
328 J.N. Jani et al. / Industrial Crops and Products 67 (2015) 324–329

prolific regeneration system. Histological examination shows the Haraguchi, H., Inoue, J., Tamura, Y., Mizutani, K., 2000. Inhibition of mitochondrial
shoot bud originates from pericycle of root. The positive effect of lipid peroxidation by Bakuchiol, a meroterpene from Psoralea corylifolia. Planta
Med. 66, 569–571.
phloroglucinol in shoot bud development and rooting also was Hoshino, Y., Mii, M., 1998. Bialaphos stimulates shoot regeneration from hairy
observed. This protocol can be immensely helpful for genetic roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium
manipulation of this plant for metabolic engineering. rhizogenes. Plant Cell Rep. 17, 256–261.
Jain, R., Sinha, A., Jain, D., Kachhwaha, S., Kothari, S.L., 2011. Adventitious shoot
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Author’s contribution coagulans (Stocks) Dunal. Plant Cell Tiss. Org. Cult. 105, 135–140.
James, D., 1979. The role of auxins and phloroglucinol in adventitious root
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SKJ helped in the formulation of the research and manuscript James, D., Knight, V., Thurbon, I., 1980. Micropropagation of red raspberry and the
preparation, JNJ carried out the work, DSN helped JNJ during cul- influence of phloroglucinol. Sci. Hortic. 12, 313–319.
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