JARS v58n2 - In Vitro Propagation of an Endangered Sikkim Himalayan Rhododendron (R. maddenii) from Cotyledonary Nodal Segments

In Vitro Propagation of an Endangered Sikkim Himalayan Rhododendron (R. maddenii) from Cotyledonary Nodal Segments
Sandeep Kumar, K. K. Singh and L. K. Rai
G.B. Pant Institute of Himalayan Environment & Development
Sikkim Unit, P.O. Tadong, Gangtok, Sikkim

Synopsis
A micropropagation method was developed for R. maddenii from the cotyledonary nodal segments of 7-week-old seedlings. Multiple shoots were initiated on Anderson medium containing a growth regulator with additives. Size of the nodal explant was an important factor in producing multiple shoots; the smaller explants could initiate more multiples. Maximum multiplication of shoots was observed on Anderson medium containing 7.0 mgl ^-1 2iP (a plant growth regulator) without agar along with additives. Shoots regenerated from nodal explants required 0.2 mgl ^-1 IBA (an auxin) for rooting. In vivo transplants grew best on peat moss; 80 percent survival was recorded.

Introduction
The genus Rhododendron is represented by fifty species in India and about 98 percent of the Indian species are found in the Himalayan region. Out of this 72 percent are found in Sikkim Himalayan region (9, 4, 19). It has been observed recently that the rhododendrons of the region are under pressure from various major quarters which are basically manmade, and some of the species, for example R. leptocarpum , R. maddenii , R. niveum , are under serious threat of extinction (20). The major threats to rhododendrons in this region are direct, such as its use as fuel wood substitute and incense, as well as indirect, such as habitat disturbances brought about by forest clearances, construction works (built-up area) and tourist influx. These interferences later pool up to bring about more nuisance to the rhododendron growing area in the form of avalanche, unchecked rains and surface flow, flashfloods, etc. The regeneration status in the form of available seedlings/saplings is very poor due to the above situation for many of the rhododendrons. The above situation could be detrimental for a selected few rhododendron species which are naturally found in small isolated populations.
Though floristically rich, the genus R hododendron in the region is one of the most neglected groups of plants in terms of scientific inquiry so far. There has been no substantial effort even to estimate the total number of species, sub-species and varieties of rhododendrons to date.
Rhododendron maddenii is a beautiful and endangered rhododendron that has limited distribution. It is the only species found in the region which produces fragrant flowers. The type species was recorded from Choongthang in Sikkim and collections were also made from Chandebi, Gyasa and Phobisika in the Bhutan Himalaya where it was reported scarce by Ludlow and Sheriff. It was also recorded at Rate-chu catchment (2000 m), epiphytic on trees. It grows in its type habitat in Choong-thang at the confluence of the rivers Lachen and Lachung on steep, rocky, precarious slopes and thickets (21).
Douglas (6) in 1984 described a method for propagation of eight cultivars of rhododendrons in vitro using agar-solidified and liquid media. Direct rooting of shoots in vivo using expanding bud explants were done on media used for propagation containing macro-nutrients, sucrose, and adenine sulphate as per Andersons formulation, the micronutrients as per Murashige and Skoog medium, and vitamins according to Gamborgs B5 formulation containing 2iP [6-(γ,γ-Dimethylallylamino) purine, a growth regulator] and IAA (Indole-3-Acetic Acid, a growth-inducing plant hormone usually effective for root promotion). Woody plants often secrete substances into the medium in response to wounding or excision often inhibiting the growth and development of explant in vitro (15). There is an important need to conserve the species through establishment of in vitro protocols and plantlet regeneration which is described in the article.

Materials and Methods
Mature seeds of Rhododendron maddenii were collected from Choongthang and Rate-chu between November-December. The explants were washed with liquid detergent Tween-80 (1ml/20ml distilled water) for five minutes, detergent removed by repeated distilled water washes and the surface disinfected with 90% ethanol (v/v, 30 sec). Some were treated with 0.15% mercuric chloride (HgCl ₂ ) for 3 minutes. Excess HgCl ₂ was removed by repeated washing with sterilized distilled water (4 or 5 times at five minutes interval) under aseptic conditions. The explants were germinated on hormone-free Murashige-Skoog (MS) medium (17; Table 1).

From 7-week-old seedlings, cotyledonary nodal portions were excised and kept for shoot regeneration in Anderson media (1) containing IAA (0.1mgl ^-1 ) and 2iP (0.5-15 mgl ^-1 ) along with the auxin TDZ [Thidiazurone (N-phenyl-N-(1,2,3-thiadiazol-5-yl)) urea], another plant growth regulator at 0.5-5.0 mgl ^-1 . Both the media were adjusted for additives, such as PVP (polyvinyl pyrrolidone) at 100 mgl ^-1 as antioxidant, ascorbic acid 100 mgl ^-1 and citric acid 10 mgl ^-1 . The pH of the media was adjusted at 5.6 prior to the addition of agar (0.8% w/v) and autoclaving at 121C for 15 minutes at 1.05 kg/cm ² pressure. The cultures were incubated under white fluorescent light 60 μmol. m ^-2 s ^-1 photon flux, 16-hour photoperiod at 171C temperature and 60% relative humidity. The shoots induced from the cotyledonary nodal portions were excised after 5-6 weeks and kept horizontally in Anderson medium containing 2iP (3.0-9.0 mgl ^-1 ) with agar and without agar (filter paper bridge) along with the same additives used for multiplication of shoots. The cultures were monitored daily and the readings were recorded after 4 weeks.

The shoots of 2-4 cm length were isolated from the shoot clump and kept for rooting in concentration gradient of Anderson medium along with growth regulating hormones, e.g., IAA, IBA (Indole-3-Butyric Acid) and α-NAA (α-naphthalene acetic acid, all at 0.05, 0.10, 0.20, 0.30 and 0.50 mgl ^-1 ). Anderson medium containing 0.20 mgl ^-1 IBA (optimum concentration) was used for filter paper bridge technique using liquid medium. Data were recorded during successive passages. The plantlets were taken out of the culture vessels after 5-7 weeks, transferred to pots (125 ml) containing autoclaved garden soil:SOILRITE (1:3) enriched with Anderson nutrient salts. The plants were kept in pots covered by polyethylene sheets and were irrigated after 4 days (Anderson nutrient salts). The polyethylene sheet was removed by gradually exposing the plantlets to culture room condition and kept in a moist saturated miniature greenhouse having 60-80% humidity. After 2-3 months the plantlets were transferred to pots containing fresh peat moss and soil (pH 4.0-5.5) for further growth under nursery conditions.

Results
The main objective of the present work was to develop a micro-propagation protocol for Rhododendron maddenii through cotyledonary nodal portion. The size of nodal explants was found to play an important role in initiation, multiplication and elongation of shoots. The smaller (0.5 cm) explants could initiate more multiples (five in four weeks). 2iP (5.0 mgl ^-1 ) with IAA (0.1 mgl ^-1 ) was found to be the best plant growth regulator (PGR) combination for initiation of culture (Table 1). In the present study in the presence of IAA no shoots emerged. The use of TDZ was also not very effective, causing browning. This problem could be solved to some extent by addition of antioxidants such as PVP, ascorbic acid and citric acid in the medium.

All the shoots exhibited continued multiplication and elongation on Anderson medium containing some additives with 2iP (7.0 mgl ^-1 ) along with agar and without agar (filter paper bridge). A monthly record on the length, number of shoots on both medium, the regenerated shoots, etc., were made.
Highest yield of shoots was obtained with 7.0 mgl ^-1 2iP liquid medium. This treatment gave a mean of 22 shoots per shoot cultured. By comparison, similar shoots cultured on agar-solidified medium with 7.0 mgl ^-1 2iP gave only 15 shoots per shoot cultured (Figure 2). In filter paper bridge media containing 7.0 mgl ^-1 2iP was the most effective concentration followed by other concentrations. Within two weeks, average of 22 shoots, having 3-5 cm length with 4-8 nodes, developed (Figure 3). Although the average shoot number and length was slightly better on these concentrations, the health and the growth of the plants and leaves were better when cultured on 5.0-9.0 mgl ^-1 2iP (Table 2). Juvenile shoots from tissue culture may be related to their more juvenile physiology and their capacity to absorb supplied nutrient and auxin during pre-treatment.

Table 1. Effects of cytokinin combined with IAA on shoots proliferation in cotyledonary nodes of R. maddenii .
A M + Plant growth regulators (mgl -1 )			Mean number of shoots SD	Mean shoot length cm SD	Callus intensity
2iP	IAA	TDZ
0.5	0.1	--	1.8 0.18	1.5 0.36	++
1.0	0.1	--	1.8 0.16	1.7 0.28	++
3.0	0.1	--	2.0 0.28	2.0 0.22	+
5.0	0.1	--	4.3 0.92	2.7 0.59	+
7.0	0.1	--	4.1 0.91	2.5 0.69	+
9.0	0.1	--	3.7 0.63	2.2 0.55	-
11.0	0.1	--	3.6 0.84	1.9 0.62	-
13.0	0.1	--	2.3 0.16	1.9 0.45	-
15.0	0.1	--	2.0 0.12	1.4 0.23	-
--	0.1	0.5	2.7 0.24	1.8 0.33	+
--	0.1	1.0	2.2 0.48	1.8 0.36	++
--	0.1	2.0	2.4 0.55	2.0 0.19	+
--	0.1	3.0	2.8 0.69	2.2 0.58	+
--	0.1	4.0	2.2 0.25	1.6 0.25	-
--	0.1	5.0	2.0 0.31	1.8 0.41	-

Table 2. Effects of different concentration of 2iP on shoot multiplication of R. maddenii in liquid and agar-solidified Anderson medium with additives.
2iP (mgl -1 )		No. of shoots SD	Length of shoots SD (cm)	Percent explant sprout
Liquid	0.0	2.60 0.18	0.8 0.24	16.0
	3.0	14.50 1.10	1.7 0.39	62.0
	5.0	16.50 1.10	2.9 0.26	78.0
	7.0	22.50 2.50	4.0 0.96	91.4
	9.0	15.20 0.80	2.6 0.41	63.0
Agar	0.0	2.10 0.16	0.7 0.20	12.2
	3.0	7.80 1.00	1.8 0.28	38.0
	5.0	12.00 0.90	2.6 0.24	42.0
	7.0	15.80 1.6	2.8 0.28	48.0
	9.0	11.80 1.50	2.0 0.18	32.0

Table 3. Effect of different auxins incorporated in Anderson medium on rooting regenerated shoots of R. maddenii after five weeks.
Auxins (mgl -1 )	Rooting (%)	No. of roots (no. SD)	Callusing
Control	22	1.50 0.57	C++
IBA
0.05	18	3.00 1.00	C++
0.10	20	3.66 0.58	C++
0.20	48	7.00 3.00	C+
0.30	36	4.66 1.15	C++
0.50	24	2.33 1.52	C+++
IAA
0.05	--	--	C++
0.10	22	5.00 1.00	C+
0.20	27	7.00 2.64	C++
0.30	19	4.00 1.00	C+++
0.50	26	3.66 1.50	C++
α-NAA
0.05	21	3.33 0.57	C++
0.10	24	4.66 2.15	C++
0.20	28	5.00 1.00	C+
0.30	22	4.66 2.08	C+++
0.5	--	--	C+++

The growth hormones , e.g., α-NAA and IAA, were ineffective at all concentrations employed in inducing rooting, whereas the IBA could induce rooting response in 48% shoots (Table 3). There was a differential requirement of IBA, i.e., 0.2 mgl ^-1 and 0.3 mgl ^-1 by the nodal explants derived shoots for the best rooting. Under this situation root initiation took more than 5 weeks on Anderson medium without agar (Figure 4). The roots were thick and healthy and new shoots continued to regenerate from the rooted basal portion of the plant on prolonged culture. Roots produced on α-NAA and IAA were small and thin along with callus formation. These plants were ready to be taken out to in vivo condition within 6 weeks from the day of inoculation. During hardening, about 12% plants died and 88% survived (Figure 5). Plantlets produced using standard protocol were subjected to hardening and acclimatization in field conditions. About 80% plants survived and only 20% mortality was recorded (Figure 6).

Discussion
The above Anderson medium with 2iP used was satisfactory for establishing culture and shoot production of Rhododendron maddenii . Variability in interaction between cultivars and medium composition was previously reported for Rhododendron (8, 16). Increased shoot production with liquid filter paper bridge supporting medium suggests that rhododendrons are capable of rapid metabolism of the medium. Previous studies using liquid media showed increased shoot production for carnation (7), Aegle marmelos (3, 22), eight cultivars of Rhododendron (6), Cammiphora wightii (13). Harris and Mason (10) suggest that mixing would reduce accumulation of toxic substance and/or depletion of essential nutrients in the immediate vicinity of shoot cultures. Alternatively, the chemical and colligative properties of agar may be detrimental.
Deberg and Maene (5) reported that cytokinin appeared less readily available to plants in agar medium as compared to liquid medium. For commercial production, liquid medium has additional advantages. Convention hardwood cuttings of Rhododendron ssp. from mature plants are difficult to root (11). In the present study, shoots gave prolific rooting in presence of IBA in 5 weeks. Optimum IBA supplied in treatment increased the number of roots per shoot. Lower concentration of IBA inefficient for rooting and higher concentration of auxins showed inhibition of roots and initiation of compact non-embryogenic callus from shoot. Supplied IBA may have increased endogenous auxin levels or level of other root-inducing substances. Alternatively, it may have reduced the relative concentration of inhibitory substances or of cytokinins (12, 13). In previous studies with rhododendrons, shoots were transplanted directly without pre-treatment, but two months were required for good rooting (14). Other workers also reported variable results and extended periods of time to achieve 70-100% rooting with direct-rooted rhododendrons from tissue culture (2, 21). Acclimatization involves the exposure of plants to reduced relative humidity and external environment without disturbing or injuring the delicate root or shoot systems. This may account for the 20% loss in transplant survival of R. maddenii . The waxy cuticle and stomata on leaves of in vitro grown plants are inadequate or inoperative which makes leaves incapable of preventing or reducing the water loss that may occur in variable humidity of the in vitro environment (18). In R. maddenii , plantlets developed after auxin, when transferred to pots, grew well with high percentage of survival in regular garden soil and peat moss mixture.

Acknowledgements
The work was supported by the Department of Biotechnology, Government of India. DBT grant No. BT/PR/1805/Agr/08/129/99. The authors are grateful to the Director, G. B. Pant Institute of Himalayan Environment and Development for providing necessary facilities and encouragement during the study.

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Dr. Sandeep Kumar, is a Research Associate, and Dr. K. K. Singh and Mr. L. K. Rai are scientists working under DBT Project "Genepool preservation and mass propagation of Sikkim Himalayan rhododendrons using biotechnological tools" at G.B. Pant Institute of Himalayan Environment and Development, Sikkim Unit, Gangtok, Sikkim, India.