Seed viability was studied for the three types of seeds, the fresh seeds collected from the plant grown in the Botanical garden, Department of Botany and Biotechnology showed 95% germination whereas, the germination 85% and 70% was observed for six months and one year stored seeds respectively. In case of six months and one year old seeds, the seeds were germinated after 22-24 days and 28-30 days respectively and 7-8 days required for the germination of fresh seeds on MS and other media also (Table 1). Out of four different strengths of MS basal media evaluated for seed germination full strength MS was found to be most effective (Table 1; Fig 1A). So, cotyledonary nodes from the fresh seeds were taken for the shoot multiplication experiment. Likewise, Vishwakarma et al., (2009), Sanatombi and Sharma (2007)
have taken fresh mature seeds of Desmodium gangeticum
and Capsicum annuum
respectively for better germination. Kambizia et al., (2006)
also reported the maximum germination in current season seeds.
Table 1: Effect of basal media strength and period of storage on seed germination.
Fig 1: A.Germinated seedling on MS, B. Multiple shoots on MS + 1.5 mg L-1 BAP in CN explant with two whole cotyledons at day 15 , C. Multiple shoots on MS + 1.5 mg L-1 BAP in CN explant with two whole cotyledons at day 30, D. Multiple shoots on MS + 1.5 mg L-1 Kin in CN explant with two whole cotyledons at day 30, E. Multiple shoots on MS + 1.5 mg L-1 BAP + 0.5 mg L-1 IAA in CN explant with two whole cotyledons at day 30, F. Rooting of in vitro shoots on 1/8 MS at day 20, G. Acclimated plantlet in 1: 1 :: sand: soil, H. M. pruriens established in earthen pots containing garden soil.
In our experiment, we have evaluated four concentrations of basal medium i.e
. MS, ½ MS, ¼ MS and ⅛ MS without any PGRs for seed germination of Mucuna pruriens
out of which MS showed highest 95% germination in 7-8 days of culture (Fig 1 A). Seed germination on MS without any PGRs was observed in Mucuna pruriens
(Faisal et al
. 2006). Similar results were observed in Arachis hypogaea, Clitoria ternatea, Desmodium gangeticum, Lathyrus ochrus, Psoralea corylifolia, Thermopsis turcica
by Venkatachalam and Kavipriya (2012)
, Mukhtar et al., (2012), Vishwakarma et al., (2009), Saglam (2012)
, Shinde et al., (2009), Cenkci et al., (2008)
respectively. Some workers like Indravathi and Pullaiah (2013)
, Parveen and Shahzad (2010)
and Barik et al., (2007)
have also used ½ MS without PGRs for seed germination in Albizia amara
, Cassia sophera, Clitoria ternatea
Multiple shoot development
PGRs are numerous chemical substances that profoundly promote and influence plant growth. Addition of various PGRs to the media promotes the growth of different plant parts. The cotyledonary nodes derived from 10 day’s old axenic seedling was experimented to various concentrations of PGRs to get the morphogenic response and the results were documented in the (Table 2). On MS medium without any PGRs the cotyledonary node showed only 01 shoot on either side. So, we have essentially added cytokinin for the induction of multiple shoots from the cotyledonary node explants. Though two different types of cytokinins (BAP and Kin) were tested, the best result was recorded with BAP. To induce multiple shoot proliferation in cotyledonary node, MS supplemented with BAP at a concentration of 1.5 mg L-1
was found to be optimum medium. The average highest (c.a.
21.1) number of shoots was found per explants (with maximum average shoot length 4.8 cm) with a frequency of 100% (averaging) at 30 days on the optimum medium (Fig 1B,C) whereas, MS with addition of Kin showed poor response in comparison to BAP. Among different concentrations of Kin, Kin 1.5 mg L-1
showed highest (c.a
. 4.8) number of shoots with 3.9 cm length (Fig 1D). Sathyanarayana et al., (2008)
obtained 16.33 no of shoots on MS + BAP 3.5 μM using axenic axillary bud which was lower than we obtained in the first harvest in M. pruriens
. Similarly, in other varieties like Mucuna pruriens
, Faisal et al. (2006)
reported ½ MS supplemented with BAP (5.0 μM) and NAA (0.5 μM) was the most suitable medium for maximum shoot induction and proliferation with 17.8 shoots with 5.9 cm length. Further our results were in accordance with the findings of Indravathi and Pullaiah (2013)
and Barik et al., (2007)
who had also reported MS + BAP 1.0 mg L-1
was the best medium for shoot culture in Albizia amara
(shoot number 6.84, length 3.20 cm) and Clitoria ternatea
(shoot number 5.2, length 6.4 cm) respectively.
Addition of auxins to the best shoot multiplication medium reduced shoot number markedly. The average number of shoots i.e
. 3.3 and 5.3 were found per explant on MS + BAP 1.5 mg L-1
+ NAA 0.25 mg L-1
and MS + BAP1.5 mg L-1
+ IAA 0.5 mg L-1
respectively (Table 2; Fig 1E). However, there was change in shoot regeneration percentage i.e.
73.3% and 80.0% in NAA and IAA respectively. BAP alone was found as the most preferred PGR for multiplication of shoots over Kin and BAP +NAA/IAA. Similar reports were observed for multiple shoot initiation in several plants including Pterocarpus marsupium
(Anis et al
., 2005), Balanites aegyptiaca (Siddique and Anis 2009)
, Terminalia bellerica
(Mehta et al
., 2012), Wrightia tomentosa
(Penchala et al
., 2015) and Canavalia gladiata (Behera et al., 2020).
Table 2: Multiple shoots regeneration from cotyedonary node with two whole cotyledons.
There is a need to speed up the shoot multiplication within less time to fulfil the purpose. Thus, after the 1st
harvest of in vitro
shoots, each mother cotyledonary node (CN) explant was sub-cultured twice where we obtained 10.2 and 4.7 shoots in 2nd
harvest respectively. A considerable decline in the shoot number during the 3rd
harvest imposed us for no more subculture of the primary explants (Fig 2). Similar observation was observed by Moharana et al. (2017)
while working on CN of Lawsonia inermis
and Behera et al., (2020)
in Canavalia gladiata
. Contrary to our results, the highest shoot number reported in the 2nd
harvest as compared to 1st
harvest and then consequently it decreased gradually during 3rd
harvest in Acacia sinuate
(Vengadesan et al
., 2002). In vitro
nodal segments obtained from the primary shoots of mother cotyledonary node were used as explants source (data not given) for further shoot multiplication. This facilitated the production of average 84.4 (c.a.
21.1 shoots/CN × c.a.
02 in vitro
nodes/shoot × c.a.
02 shoots / in vitro
nodes) number of shoots per in vitro
cotyledonary node after the 1st
harvest at day 30 whereas, a total c.a.
144 shoots (c.a.
) were harvested after 3rd
culture of the mother cotyledonary node with two whole cotyledons at day 70 (30 + 20 + 20; Fig 2). In our case, we reported higher number of shoots while Suresh et al., (2015);
reported only 1-2 shoot regenerated from axenic node and Ozaki et al., (1993)
reported one shoot from immature leaflet of 10 d old axenic seedling. So far, a single report for in vitro
micropropagation of M. pruriens
using cotyledonary node explants was reported by Sathyanarayana et al., (2008),
obtained 16.33 no of shoots on MS + 3.5 μM BAP which was lower than what we obtained in this experiment.
During the experiment different forms of the cotyledonary node (CN with two whole cotyledons, CN with two proximal halves, CN with one whole cotyledon and CN without cotyledon) were taken into consideration for shoot multiplication because number and size of cotyledons also influenced the shoot regeneration capacity (Moharana et al., 2017).
It was observed that shoot regeneration frequency percentage remained the same with a variation in shoot number and length (Fig 2). Highest shoot number of 21.1 and 3.1 with average shoot length of 4.8 and 1.8 cm resulted in CN with two whole cotyledons and CN without any cotyledon respectively on MS + 1.5 mg L-1
(Fig 2). The results were in accordance with findings in grass pea (Barik et al., 2004),
blackgram (Das et al., 1998)
and mungbean (Sen and Mukherjee 1998
). Complete removal of both cotyledons caused a delayed shoot regeneration response and produced fewer shoots. Similar observations were also reported in mung bean (Gulati and Jaiwal 1994
; Polisetty et al., 1997)
and sword bean (Behera et al., 2020). Saglam (2012)
used longitudinally sliced half CN (maximum 5.92 shoots) in Lathyrus ochrus,
while Vidoz et al., (2012)
used longitudinally cut cotyledons along the midrib in Lotononis bainesii
, to get the best responses.
Rooting and acclimatisation of in vitro regenerated shoot
Fig 2: Influence of cotyledon in cotyledonary node on shoot multiplication during different harvest periods. TWC: CN with 02 whole cotyledons, TPHC: CN with 02 proximal halves, SWC: CN with 01 whole cotyledon and WC: CN without cotyledon.
Rooting of in vitro
regenerated shoots was experimented on five different concentrations of MS medium and out of those 1/8 MS medium devoid of any PGRs responded faster in terms of the day and facilitated better rhizogenesis. Root induction was started within 5-6 days and in 14-15 days the average root number and length were 7.0 and 5.4 cm respectively (Table 3; Fig 1F). Though ¼ MS and ⅛ MS gave same response at a time but later ⅛ MS was found to be the optimum medium in terms of root number and length. MS and ½ MS responded slower than the above mediums followed by 1/16
MS and resulted in less number of roots. In our experiment, it seems as if PGRs is not essential for rooting. Likewise, another species of Fabaceae i.e
. Psoralea corylifolia,
developed maximum rooting (highest number 5.4 roots per explant) and highest length 10.1 cm in MS without any PGRs (Shinde et al., 2009).
But in Mucuna pruriens
, Faisal et al., (2006)
found satisfactory improvement in rooting on ½ MS medium containing IBA 2.0 mM with good length 5.5 cm and number 7.8 of roots per shoots. Luna et al., (2003)
also developed maximum number of roots in Ilex dumosa
in ¼ MS medium when supplemented with IBA 7.3 mM.
Table 3: Rooting of in vitro shoots.
Plantlets with leaflets and well-developed roots were successfully acclimatized by transferring it to plastic glasses containing soil and sand in 1:1 and eventually established in pots containing garden soil (Fig 1G, H). The survival rate of the plantlets after transfer to soil:sand (1:1) was 100% and 58.33% of the plants transferred to garden soil survived. To acclimatize the in vitro
rooted plantlets of Mucuna pruriens,
Faisal et al.
(2006) transferred them to the plastic pots filled with sterile soilrite and 90% of plantlets survived successfully. In Sinningia speciose, Kashyap and Dhiman (2011)
have acclimatized the in vitro
raised plantlets by taking four different hardening media, out of which sand and soil in 1:1 showed 45.16% survival rates. Venkatachalam and Kavipriya (2012)
used Soil and sand in 2:1 for successful acclimatization in Arachis hypogaea.
In this study, the protocol developed for Mucuna pruriens
using cotyledonary node as explants found to be most efficient reproducible and reliable one which can be used for the large-scale production and conservation of the elite variety.