REGENERATION COMPETENCE OF AN ORNAMENTALLY IMPORTANT

EPIPHYTIC ORCHID, RHYNCHOSTYLIS GIGANTEA (LINDL.) RIDL.

THROUGH LEAF SEGMENTS: A STUDY IN VITRO
Promila Pathak, Shivani Verma, Ankush Prakash, and K C Mahant1

Orchid Laboratory, Department of Botany, Panjab University, Chandigarh-160 014, U.T., India
1
Goverment Post Graduate College, Nalagarh, Distt. Solan, Himachal Pradesh, India

Abstract

This paper elucidates the possibility of using leaf segments for micropropagating Rhynchostylis gigantea. Mitra et al. (1976, M) medium
supplemented with KN (1.5 mgl-1) proved optimal nutritional combination for initiation, multiplication, and early plantlet formation in Rhynchostylis
gigantea leaf culture. The plantlets, thus raised were subjected to hardening procedure (in vitro and ex vitro) and were established with
about 70% survival frequency.

Introduction Mitra et al. (1976, M) medium without and with different

concentrations and combinations of PGRs [( IAA, NAA,
RHYNCHOSTYLIS GIGANTEA (Lindl.) Ridl., commonly BAP, KN, IAA+KN, IAA+KN, NAA+BAP) see table1].
known as Fox tail Orchid is widely distributed in India The cultures were incubated in the ambience of 25±20C
(tropical Himalayan valleys from Sikkim Westwards to and 12 hr photoperiod of 3,500 Lux light intensity. The
Garhwal and Eastward to Bhutan), Myanmar, Thailand, results were analyzed using one way analysis of
Malaysia, Vietnam, China, Bangladesh and the variance performed with respect to each response
Philippines. The species is well known for its beautiful (average ± standard error) against each additive. As
white flowers in long compact and pendant ANOVA results showed the non significant difference
inflorescences. Due to extensive habitat destruction of additives at 5% level of significance, various groups
and commercial collection pressure, its natural of additives showing identical/similar response were
populations are on decline (Rittirat et al., 2011). The formed statistically. To this end, Tukey Test was
species of economic importance, figures prominently performed at 5% level with respect to each response.
on the list of rare plants of India. Hence, propagation of
the species is urgently required with a view to Results
conserving the species. Earlier, though the regeneration
potential of various explants (seeds, stem, leaf, root, In the present study, entire leaves from axenic sources
inflorescence etc.) has been tested in vitro in different were used for regeneration of Rhynchostylis gigantea.
species (Anuprabha and Pathak, 2012; Arora et al., The regenerative competence of foliar explants of the
2014, 2016; Bhattacharjee and Hossain, 2015; Borah species was significantly influenced by the nutrient
et al., 2015; Chauhan et al., 2010, 2015; Hegde, 2012; medium, quality and quantity of PGRs, segmentation
of leaf explants and orientation of explants on the
Hoque et al., 2016; Kaur and Pathak, 2014; Laqishram
medium. Similar results were obtained earlier by Deb
and Devi, 1999; Pathak et al., 1992, 2011, 2012, 2016;
and Pongener, 2013 and Pathak (1989). According to
Sibin and Gangaprasad, 2016; Sibin et al., 2014; Verma
Wimber (1965), PLBs from the leaves of cymbidiums
et al., 2013; Vij et al., 1987,1989, 1994, 1995) so as to
were successfully developed which opened up an
develop protocols for their in vitro propagation, the data
effective alternative to apical shoot meristem cultures.
is, however, meager in terms size of the orchid family.
The regeneration competence of leaf explants was
Presently, an attempt was made to develop an appropriate positively tested for more than 60 orchid species and
in vitro propagation method for Rhynchostylis gigantea, success is restricted mostly with epiphytic orchids and
using entire leaf segments. Some of the important only a few species from terrestrial orchids have
features of the study are documented in this paper. responded for he purpose (Deb and Sungkumlong, 2010).

Materials and Methods Preently, the regeneration potential of whole leaf

segments (0.5-1 cm long) procured from 26 wks old in
Whole leaf segments (0.5-1 cm long) obtained from 26 vitro raised cultures was successfully tested using Mitra
wks old in vitro grown cultures were assessed using et al. (1976, M) medium with and without different
Received: May 5, 2016; Accepted: December 05, 2017
Table1. Regeneration pot ential of in vitro sourced leaf explants (entire) in Rhynchostylis gigantea on Mit ra et al. (1976, M) medium.
J. ORCHID SOC. INDIA

Entries in column nos. 2, 3 and 7 are Mean ± S.E.; same alphabetical letter in the superscript denotes that the corresponding means are in the same group using Tukey test at 5%.
(DECEMBER 30,
2017) PATHAK ET AL. - LEAF SEGMENTS AND REGENERATION

Figs. 1-13. In vitro leaf explant (entire) culture of Rhynchostylis gigantea: 1, Formation of PLBs at the base of shoot [M+IAA (1.5 mgl -1)]; 2,
Direct rooting at the base of leaf [M+IAA (2 mgl-1)]; 3, Formation of multiple leaves [M+BAP (2mgl-1)]; 4-5, Multiplication of PLBs and early
complete plantlet [M+KN (1.5 mgl-1)]; 6-10, Formation of complete plantlet [M+KN (2mgl-1), M+IAA (1 mgl-1)+KN (2 mgl-1)], [M+IAA (1 mgl-
1
)+KN (2 mgl-1)], [M+ IAA (2 mgl-1)+KN (1 mgl-1)]; 11, Plantlets with long, thick and healthy roots and complete plantlet formation [M+NAA (1.5
mgl-1), NAA(2 mgl-1)]; 12, Additional shoot buds at the base of shoot [M+NAA (1mgl-1) +BAP (2mgl-1)]; 13, Seedlings transferred to plastic
pots.

combinations and concentrations of PGRs (IAA, NAA, within 5 wks of inoculation. In the present study, initiation
BAP, KN, IAA+KN, IAA+KN, NAA+BAP; Figs. 1-13). of morphogenetic response was restricted to the basal
The leaf segments failed to respond in the basal region of the leaf. The morphogenetic potential of leaf
medium; these turned brown and subsequently perished base has also been reported in several other species
 J. ORCHID SOC. INDIA (DECEMBER 30,

such as Arachnis labrosa (Deb and Temjensangba, well documented in orchids (Arditti and Ernst, 1993;
2007) and Vanda coerulea (Vij and Aggarwal, 2003). Deb and Sungkumlong, 2010; Deb and Temjensangba,
Mathews and Rao (1985) considered the leaf base to 2007; Li and Xu, 2009; Temjensangba and Deb, 2005;
be the decisive factor for culture initiation from foliar Vij and Pathak, 1990; Yam and Weatherhead, 1991).
plants. Murashige (1974) opined that in vitro plant regeneration
occurs frequently through adventitious shoot formation
IAA when used at 1.5 mgl-1 in the medium induced initiation and rarely through somatic embryogenesis (Deb and
of regeneration response in 50.00±0.81% segments in Pongener, 2013). Presently, regeneration also occurred
30.00±0.81 days via shoot bud formation; 5 additional via PLBs formation, in accord with similar earlier
PLBs were activated at the base of shoot (Fig. 1) and observations in case of Dendrobium and Epidendrum
plantlets were developed within 124.00±0.81 days. (Churchill et al., 1970, 1971), where shoot regeneration
Interestingly, direct rooting was induced at the base of from leaf explants has been reported to occur through
the whole leaf when IAA was used at higher concentration the formation of PLBs.
(2 mgl-1) in the nutrient pool (Fig. 2). NAA when used at
lower concentration (1.5mgl-1), 50.00±0.81% segments Acknowledgement
responded in 48.75±0.95 days, 2 meristematic loci were
activated and plantlets with bright green leaves and long, Financial assistance from University Grants
healthy and thick roots were obtained in 60.00±0.81 days Commission, Delhi, India is gratefully acknowledged.
(Fig. 10). NAA when used at higher concentration
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