in vitro rooting efficiency in morus indica cultivars (s34, s54, m5...

Current Trends in Biotechnology and PharmacyVol. 8 (3) 288-293 July 2014, ISSN 0973-8916 (Print), 2230-7303 (Online)

288

AbstractDevelopment of plant tissue culture has

contributed greatly to our understanding of thefactors responsible for the growth anddifferentiation of plant cells which resulted inregenerating whole plants from the culturedmaterial. Mulberry plant is usually heterozygousdue to its cross pollination habit. Use ofmicropropagation as an integral component oftree improvement program has lagged behind forsome important forest tree crops because of thefailure to develop protocols for successful rootinduction. An attempt has been made to describean efficient root induction protocol from in vitroregenerated shoots of all the mulberry cultivars(cvs. S34, S54, M5 and Mysore-local) under study.Efficient rooting 92% response was observed onMS media containing IBA with 1.0mg/l and 61%of rooting response noted on mediacontaining1.0mg/l IAA with mulberry cultivars (cvs.S34). The rooting ability was found to be superiorin cvs. S34 when compared to other cultivars ofmulberry.

Keywords: Murashige and Skoog, Indole-3-Butyric acid, Indole acetic acid, Cultivar (cv.).

Introduction One of the essential requirements for thesuccessful application of plant propagationtechnology to agriculture is the capacity toregenerate elite plantlets. The successfuloutcome to micropropagate plantlets through

tissue culture essentially depend on induction ofrooting. Mulberry leaves show the synthesis ofpharmaceutical and commercial products suchas the sericin protein consumed by Bombyx moriyields cocoons. The leaf biomass producesenergy rich substances like vitamins A and D, alsohelps in prevention of certain gastric disorders(1). Any means of micropropagation leads tosuccess only on establishing efficient rootinduction protocols in tissue culture (2,3).Micropropagation as an important component ofcrop improvement systems has lagged behind forsome significant tree crops because of the failureto develop techniques for successful root initiation(4,5). Problems encountered during rooting of invitro regenerated shoots are mainly related to thephenomenon of callus induction from the cut endsof the shoots, when high auxin concentration isusually applied for root initiation (6). Here wedescribe efficient root induction (7) anddevelopment protocol from in vitro regeneratedshoots (8,9) of all the mulberry cultivars. Our studydemonstrates the rooting ability to be superior incv. S34 (Plate -I Fig. a-d) compared with othercultivars (M5, Mysore-local and S54) under study.

Materials and Methods Nodal explants of four mulberry cvs. namelyM5, S34,S54 and Mysore-local were collectedfrom 3-4 months old field grown mature plants(10). Explants were surface disinfected with 0.1% HgCl2 for 3-5 minutes and followed by 4-5rinses in sterile distilled water. The explants were

In vitro rooting efficiency in Morus indica cultivars(S34, S54, M5 and Mysore-local) from in vitro shoot

cultures

Lavanya Kakarla and Chakravarthy Rama*Department of Biotechnology, KLUniversity, Vaddeswaram, Guntur district – 522502,

Andhra Pradesh. India.*For correspondence: [email protected]

In vitro rooting efficiency in Morus indica cultivars


289

4 weeks of culture (13). Percentage of rootedplants, average number of roots per shoot androot length was recorded in all the cultivars (S34,M5, Mysore-local and S54) after 20-25 days inculture.

Results and Discussion

Effect of IBA: Effect of IBA on in vitro rootdevelopment and the percentage of response,average number of roots and average root lengthin all the mulberry cultivars were studied.Regenerated shoots (14, 15, 16) from all thecultivars responded with a high rooting efficiencyat 1 mg/l IBA in cv. S34 (92%) (Fig-2(a)) followedby cvs.M5 (80%) (Fig-2(b)), Mysore-local (72%)(Fig-3(b)) and S54 (60%) (Fig-3(a)). However at0.5 mg/l IBA there was no significant responsewhen compared with 1 mg/l IBA. The averagenumber of roots produced at 0.5 mg/l IBA wasless when compared to higher concentrations (1.0– 1.5 mg/l) of IBA.

In vitro regenerated shoots of cvs. M5, S34,S54 and Mysore-local were cultured on MSmedium supplemented with differentconcentrations of IBA (0.5 – 2.0 mg/l). From theabove concentrations 1 mg/l IBA was reported tobe optimum for root induction in all the cultivarsstudied.

Fig. 1(a-d). Efficient rooting performance recordedfrom in vitro regenerated shoots in mulberry cv.S34on MS + 1 mg/l IBA.

Fig. 2. (a). Root initiation from in vitro regeneratedshoots on MS + 1 mg/l IBA in mulberry cv. S34. (b).Root induction on MS + 1 mg/l IBA from in vitro grownshoots in mulberry cv.M5

Fig. 3. (a). Root initiation from in vitro regeneratedshoots on MS + 1 mg/l IBA in mulberry cv. S54. (b).Root induction on MS + 1 mg/l IBA from in vitro grownshoots in mulberry cv.Mysore-local.

cultured on MS medium supplemented withdifferent concentrations (0.5-2 mg/lit) of BA or KNand combinations of BA (1.0 mg/lit) and KN (0.5-2 mg/lit). The pH was adjusted to 5.7 and solidifiedwith 0.8% difco bacto agar. Single explants wereinoculated in each culture tube and incubated at25 ± 2 °C for 2-3 weeks under 16 hoursphotoperiod.

In vitro regenerated shoots were rooted onfull strength MS medium (11,12) supplementedwith auxins like IAA or IBA (0.5 - 2.0 mg/l) after 3-

Lavanya Kakarla and Chakravarthy Rama


290

The average number of roots were foundto be maximum in cv. S34 (19.2 ± 0.3) (Table-1,)followed by cvs. M5 (13.6 ± 0.2) (Table-2 ), Mysorelocal (9.6 ± 0.2) (Table-3 ) and S54 (8.2 ± 0.2)(Table- 4 ) at 1 mg/l IBA. There was a gradualdecrease in the average number of roots withincrease in concentrations (1.5 – 2.0 mg/l) of IBA.Maximum mean length of the root was recordedin cv. S34 (6.5 ± 0.6) followed by cvs. M5 (4.5 ±0.4), Mysore-local (3.2 ± 0.4) and S54 (2.2 ± 0.2)at 1 mg/l IBA. At higher concentrations of IBA (1.5– 2.0 mg/l) showed a gradual decrease in themean length of the root in all the cultivars studied.Moreover, 0.5 mg/l IBA was reported to be leastfavorable for root induction.

Effect of IAA: Effect of IAA on in vitro rootdevelopment, percentage of response, averagenumber of roots and average root length was alsostudied. Regenerated shoots cultured on rootingmedium showed highest percentage of rooting incv. S34 (61%) (Fig-4 (a)) followed by cvs. M5(48%) (Fig-4 (b)), Mysore-local (40%) (Fig-5(b))and S54 (32%) (Fig-5(a)) at 1mg/l IAA.Percentage of response was poor at 0.5 mg/l IAAand was less significant when compared to higherconcentrations of IAA (1.5 and 2.0 mg/l).

The average number of roots grown werefound to be high in cv. S34 (9.6 ± 0.3) (Table-1)followed by cvs. M5 (9.2 ± 0.2) (Table-2 ), Mysorelocal (7.8 ± 0.2) (Table-3 ) and S54 (6.0 ± 0.2)(Table-4 ) at 1 mg/l IAA. There was a considerabledecrease in the average number of rootsproduced at higher concentrations (1.5 and 2.0mg/l).

The mean length of the root was maximumat 1.0 mg/l IAA in all the mulberry cultivars studied.It was found to the maximum in the cv. S34 (5.7 ±0.6) at followed by cvs M5 (3.0 ± 0.4), Mysore –local (2.2 ± 0.1) and S54 (1.4 ± 0.1) at 1 mg/l IAA.There is a gradual decrease in the mean lengthof the root at higher concentrations (1.5 & 2.0mg/l).

The study is conducted to mark the rootingefficiency and also to determine the effect ofauxins on rooting performance of in vitro raised

mulberry plants. It was found that 1 mg/l IBA wasmore favorable in inducing in vitro rootdevelopment in mulberry. The size and length ofthe root were variable in different mulberrycultivars. Root length is maximum in S34 and M5followed by Mysore local and S54.

The thickness of the root in S34 and M5were more when compared with Mysore-local andS54. At 0.5 mg/l IBA thickness was found to beless, but it increased when higher concentrations(1.5 and 2.0 mg/l IBA) were used. Roots showed

Fig. 5. (a). Root initiation from in vitro regeneratedshoots on MS + 1 mg/l IAA in mulberry cv. S54. (b).Root induction on MS + 1 mg/l IAA from in vitro grownshoots in mulberry cv. Mysore-local.

Fig. 4. (a). Root initiation from in vitro regeneratedshoots on MS + 1 mg/l IAA in mulberry cv. S34. (b).Root induction on MS + 1 mg/l IAA from in vitro grownshoots in mulberry cv.M5.



291

positively phototropism at the early stages ofdevelopment. Root induction was extensive interms of length, thickness and size at 1.0 and 1.5mg/l IBA and was not much significant at 0.5 mg/

l IBA. The highest root number was recorded inS34 followed by M5, Mysore local and S54. Effectof IAA is poor when compared to IBA in the rootinitiation from in vitro regenerated shoots of

Table 1. Effect of different concentrations of IBA and IAA individually on rooting performance of in vitro grown shoots in mulberry cv. S34.

Growth No. ofregulators Cultures % of Avg.no. Avg. root(mg/l) Maintained response of roots Length

IBA0.5 25 15 2.6 ± 0.1 0.82 ± 0.091.0 25 92 19.2 ± 0.3 6.5 ± 0.601.5 25 55 7.6 ± 0.2 2.1 ± 0.102.0 25 28 4.0 ± 0.2 1.1 ± 0.10

1AA0.5 25 9 2.0 ± 0.06 0.68 ± 0.081.0 25 61 9.6 ± 0.3 5.7 ± 0.601.5 25 46 6.8 ± 0.2 1.6 ± 0.102.0 25 17 3.2 ± 0.09 0.96 ± 0.09

Values are mean of 25 explants ± S.E

Table 2. Effect of different concentrations of IBA and IAA individually on rooting performance of in vitro grown shoots in mulberry cv. M5.


IBA0.5 25 12 2.1 ± 0.1 0.74 ± 0.081.0 25 80 13.6 ± 0.2 4.5 ± 0.401.5 25 48 6.4 ± 0.2 1.9 ± 0.102.0 25 22 3.6 ± 0.2 0.98 ± 0.10

1AA0.5 25 6 1.6 ± 0.04 0.46 ± 0.061.0 25 48 9.2 ± 0.2 3.0 ± 0.41.5 25 36 5.6 ± 0.1 1.4 ± 0.12.0 25 12 3.0 ± 0.09 • 0.08




292

Table 3. Effect of different concentrations of IBA and IAA individually on rooting performance of in vitro grown shoots in mulberry cv. Mysore-local.


IBA0.5 25 9 1.85 ± 0.09 0.52 ± 0.071.0 25 72 9.6 ± 0.20 3.2 ± 0.401.5 25 36 5.0 -± 0.20 1.5 ± 0.102.0 25 19 3.0 ± 0.20 0.9 ± 0.10

1AA0.5 25 4 1.2 ± 0.04 0.36 ± 0.061.0 25 40 7.8 ± 0.20 2.20 ± 0.101.5 25 28 4.2 ± 0.10 0.98 ± 0.102.0 25 8 1.5 ± 0.06 0.42 ± 0.08

Values are mean of 25 explants ± SE

Table 4. Effect of different concentrations of IBA and IAA individually on rooting performance of in vitro grown shoots in mulberry cv. S54.


IBA0.5 25 4 1.24 ± 0.03 0.38 ± 0.061.0 25 60 8.2 ± 0.20 2.2 ± 0.201.5 25 30 4.2 ± 0.20 1.2 ± 0.102.0 25 16 2.8 ± 0.20 0.85 ± 0.09

1AA0.5 25 2 1.0 ± 0.02 0.18 ± 0.021.0 25 32 6.0 ± 0.20 1.4 ± 0.101.5 25 16 3.6 ± 0.09 0.64 ± 0.092.0 25 4 1.21 ± 0.06 0.21 ± 0.02


mulberry. Studies on the use of IBA in rooting ripemulberry cuttings is a well established fact.Efficient rooting 92% response was observed onMS media containing IBA with 1.0mg/l withmulberry cultivars cvs. S34 (Fig-1(a-d)).

ConclusionAmong the different mulberry cultivars

studied cv S34 showed better response in rootingefficiency when compared to other cultivars.Efficient root induction results in better nutrient



293

absorption which in turn helps in enhancing theleaf biomass for commercial production of silkand improving other qualitative and quantitativetraits.

AcknowledgementsWe are very much thankful to the HOD, Board ofmanagement of K L University for providingfellowship and support for doing this worksuccessfully.

References1. Chinnaswamy, K.P., Doreswapy,C.,

Devaiah, M.C and Venkatesha,M. (1993).Pharmaceutical value of mulberry. Indiansilk, 31(11): 40-43.

2. Bonga, J.M., Reinert and Bajaj Y.P.S.(1981). In applied and fundamental aspectsof plant cell, tissue and Organ culture,Spinger verlag, Berlin, Heidelberg, pp. 93-108.

3. Ravindran, S., Dandin, S.B and Jain, A.K.(1988). Application of plant cell, tissue andorgan culture in mulberry improvementprogrammes. Indian Silk, (Dec.Ed), 37-39.

4. Honda T. (1970). Studies on the propagationof mulberry trees by cuttings. Bull Sericult.Exp. Sta. 24(1): 231-236.

5. Yadav, U., Madan, L and Jaiswal, V.S.(1990). Micropropagation of Morus nigrafrom shoot tip and nodal explants of maturetrees. Scientia Hort, 44: 16-67.

6. Chand,P.K., Sahoo Y and Patnaik S.K.(1994). Prospects of in vitro geneticmanipulations in mulberry sericulture. IndianSilk, 33: 3-6.

7. Obul Reddy, B., Giridhar, P andRavishankar, G.A. (2001). In vitro rooting ofDecalepis hamiltonii Wight and Arn anendangered shrub, by auxins and root-promoting agents, Current Science, 81(11): 1479-1482.

8. Chakravarthy, R and Sadanandam, A.(2003). In vitro propagation and seedlessfruit formation in nodal explant cultures ofMorus indica L. cv. S 54. Asian Journal ofMicrobiol. Biotech. Envi Sci, 5(4) : 551 -554.

9. Chakravarthy, R., Pavan, U, Srinivas Reddyand Sadanandam, A. (2001). Thidiazuroninduced organogenesis from mature leafcultures in mulberry (Morus indica L.) cv. S54. Sericologia France, 41(2) : 269 -273.

10. Hossain, M., Rahman, M., Zaman, A andJoarder, O.I. (1991). Effect of nature ofexplants and PH on in vitro propagation ofsome mulberry genotype. Bull. Sericult.Res., 2: 13-22.

11. Murashige T. (1974). Plant propagationthrough tissue culture. Ann. Rev. PlantPhysiol, 25 : 135-166.

12. Murashige T, and Skoog , F. (1962). Arevised medium for rapid growth andbioassay with tobacco tissue culture. Physiol. Plant, 15 : 473-497.

13. Biswas, S. and Sengupta, K. (1993). Effectof hormones on the mulberry – A Review.Sericologia, 33(3) : 461-478.

14. Kathiravan, K., Ganapathi, A and Shajahan,A. (1997). Adventitious shoot formation andplant regeneration from callus cultures ofmulberry (Morus alba L.). Sericologia, 37(4):727-733.

15. Kathiravan, K., Shajahan, A and Ganapathi,A. (1995). Evaluation of in vitro response

of mulberry genotypes. Sericologia, 35(2):305-317.

16. Tewary, P.K., Raghunath, M.K.,Venkateswarlu, M and Sarkar, A. (1996).Genotype differences in response to in vitroshoot development of mulberry (Morusspp.). Indian J. Seric., 35 : 104-106.


in vitro rooting efficiency in morus indica cultivars (s34, s54, m5...

Documents