JARS v62n3 - Raising Planting Materials of Sikkim Himalayan Rhododendron through Vegetative Propagation Using "Air-wet Technique"

Raising Planting Materials of Sikkim Himalayan Rhododendron through Vegetative Propagation Using "Air-wet Technique"
K. K. Singh, S. Kumar and Shanti Rai
G.B. Pant Institute of Himalayan Environment and Development, Sikkim Unit
Pangthang, Gangtok, East Sikkim, India

Summary
This study investigates the simple propagation techniques and optimal conditions for the vegetative propagation of the rhododendron species from leafy stem cuttings using air-wet technique. One-node cuttings had the same rooting ability as two-node cuttings. Winter cuttings showed better rooting capacity than autumn cuttings. Experimentation with rhododendron using an "air-wet" method has proved successful and seems to be a promising alternative and has been used effectively for rooting of Rhododendron . A method for vegetatively propagating of Rhododendron spp. is given.

The genus Rhododendron L. ( Ericaceae ) is perhaps the largest genus of woody plants in existence, with about 1000 species (Leach, 1961 and Chamberlain et al ., 1996) mostly concentrated in Eastern Himalayas, Western China, temperate North America and Malaysia. Sikkim state is a rich repository of plant diversity; a large number of plants are known for their endemism and threat status. Rhododendrons have been considered key indicators of a healthy forest ecosystems, especially along temperate zones in Sikkim Himalaya (Singh et al ., 2003). They are the most striking group of plant in terms of botanical entity as well as for aesthetics and local environment. The genus Rhododendron is represented by eighty species in India (Gamble, 1936; Cowan & Cowan, 1938; Pradhan & Lachungpa, 1990; and Bhattacharyya & Sanjappa, 2009). About 36 species of rhododendrons in Sikkim are found at heights of 3,030m. They exhibit good diversity in size and colours, some are 30-meter-tall trees, others are creeping dwarfs and some are found growing in the crowns of other trees as epiphytes. Mostly known for their beautiful flowers, rhododendrons also include plants with striking and often fragrant foliage. Rhododendron leaves can be evergreen or deciduous. The evergreen leaves vary strongly in size and shape. The deciduous leaves often have bright autumn colours. The leaves contain flavonoids and phenolic acids (Prakash et al ., 2007). The presence of phenolics in the rhododendrons make them an excellent source of firewood that burns even in wet condition. Rhododendrons in Sikkim usually grow under a moist environment with plenty of soil moisture and humidity. They usually require acidic soils, but some prefer alkaline soils. Most prefer moist but well drained soils; however, some also grow in bogs (Chamberlain, D.F. 1982 and Cullen & Chamberlain, 1978).
At present the rhododendrons of the region have reached a stage where many species have entered into threat categories. Many species with varying degrees of threat within their habitat were noted during base line assessments. Signs of pressure over the plant resources have started becoming visible in this region whereas some of the plant species are under severe pressure. Now with evidence of extraction of rhododendrons for fuelwood, the species have become an item for immediate conservation in the region. During tree-felling exercise, governmental or private, the epiphytic rhododendrons suffer greatly Pradhan and Lachungpa (1990) reported that massive landslides due to heavy rainfall in 1980 at Yakchey La, North of Lachung, North District Sikkim, resulted in the degradation of entire population of R. niveum . As the rhododendron habitats are now under recurrent pressure and are frequently violated, the genus has became a taxon of considerable conservation importance.
The regeneration status in the form of available seedlings/saplings has become very poor due to the above situation for most of the rhododendrons (Kumar et al ., 2004). The above situation could be detrimental for a selected rhododendron species which are naturally found in small isolated populations. Though floristically rich, the genus Rhododendron in the region is one of the most neglected groups of plants in terms of scientific inquiries done so far.
As a result of the low priority attached to rhododendrons, no attempts were made to raise planting materials of these species. Recently Konrad (2005) has reported stimulation of rooting in stem cuttings taken from blueberries and apples using air-wet technique. There are various methods of plant propagation, or multiplication. Propagation through new seed is the most common. However, new plants can also be created by cutting off a portion of an established plant. This "cutting" is placed in an environment that encourages it to produce new roots and/or stems, which later results in development of a new, independent plant.
This study investigates suitable techniques of raising planting materials of some rhododendron species. Air-wet techniques were conducted during the study trials on vegetative propagation by stem cutting.
The method used for propagation of planting materials by stem cutting under this experiment is based on the technique used by Mark Konrad (2005). Generally the best time for propagation of woody tree species was late winter or early spring prior to any vegetative bud break. The stem cuttings of rhododendron species were taken in late winter when active growth was arrested. Small branches were collected from R. dalhousiae , R. arboreum , R. maddenii and R. griffithianum (exact age not known) growing in the Rate-chu, East Sikkim (Longitude 27°5'47" to 28°8'50" North, and Latitude 88°56'27" to 88°56'25" East with an elevation 2500 m amsl). The preparation of cutting materials was done in the morning between 8.00 and 11.00 a.m. The final cuttings (average length 18 cm, 1.0-2.5 cm dia. with at least 1-2 nodes per cuttings) were clipped from the branches.
The semi-hardwood cuttings measuring 18 to 20 cm were wounded on the distal stems but only on the upper side and placed on moistened absorbent paper (Fig. 1A). The hardwood cutting with two to four leaves were cut to two equal halves and small squares of the absorbent paper (1½ in.) were used to cover the wounded sites and misted so that satisfactory moisture is maintained at all the times. The container was covered with Saran Wrap plastic sheeting. Hand misting is done as needed to maintain appropriate moisture levels. The container was kept under white fluorescent light 60 μmol m-2 s-1 photon flux, 16 hr photoperiod at 20 ± 1°C temperature and 60% relative humidity. After 16-24 weeks, initiation of callus and sprouting of dormant buds (Fig. 1B) were observed in all the species. After 16 weeks, when significant callusing had taken place, the cuttings were transferred to 9-inch pots containing autoclaved fresh peat moss and Solirite (1:1) in the greenhouse. Clear plastic sheeting was then laid over the potted plants to maintain high humidity until rooting. The mean monthly temperatures of the greenhouse ranged from 20°C (October) to 27°C (July). Monthly relative humidity of the greenhouse ranged from 70 to 82%. Observations were taken 24 weeks after planting for determining percent of rooting, number of roots formed and length of individual roots. Percent of rooting was estimated based on the total number of cuttings planted.

Table 1. Performance of rooted stem cutting of Rhododendron spp. using air-wet technique.
Species	Callusing	Rooting %	Mean root no. per rooted cutting ± SE	Mean root length (mm) per rooted cutting ± SE
R. dalhousiae Hook. Lady Dalhousie’s Rhododendron Nepali - Lahare Chimal	C+	50	5.3±0.19	16±0.12
R. arboreum Smith Scarlet Arborescent Rhododendron Nepali- Lali Gurans	C+++	80	5.7±0.33	33±0.72
R. griffithianum Wt. Lord Auckland’s Rhododendron Nepali - Seto Chimal Lord	C++	40	2.8±0.196	26±0.28
R. maddenii Hook. Major Madden’s Rhododendron Nepali - Major Madden ko Chimal	C+	_	_	_
LSD (p=0.05)		28.7	2.1	22.8
SE: Standard error. A dash (-) indicates that the cutting had completely dried. Cuttings with roots >1 mm were considered to have rooted and included for calculating % rooting. Root initials >2 mm were considered for calculating mean number of roots and mean root length. C+: indicates less callusing, C++: indicates more callusing, C+++: indicates profuse callusing. All values are an average of 20 replicates.

Rooting of 1-2 node cuttings was compared for R. dalhousiae , R. arboreum , R. griffithianum , and R. maddenii . One-node cuttings had the same rooting ability as two-nodal cuttings. Observations of initiation of rooting after 30 weeks were 50% in R. dalhousiae , 80% in R. arboretum , and 40% in R. griffithianum . Rhododendron maddenii resulted in poor callusing at the base of cuttings and did not survive and dried within a period of six weeks (Table 1). The mean numbers of main roots per cutting ranged from 2.8 to 5.7. The mean length of longest root ranged from 16 to 39 mm. Within 8-12 months the cuttings developed roots and leaves. All cuttings without leaves failed to root, indicating that leaf presence was essential for rooting. Different rhododendron species had the same rooting response to leaf presence on cuttings. Well-developed rooted plants were transferred in thermocole cups containing a mixture of autoclaved fresh peat moss and soil (1:3) and were placed under high relative humidity (80%) in the mist chamber of a greenhouse (25°C). After one month these were planted in polythene bag containing normal garden soil; all rooted stem cutting survived under green house conditions (Fig. 1C, D, E).

This is the first report for rooted stem cutting of Sikkim Himalayan rhododendron species where plants have been successfully produced and transferred to the field. It can be concluded from the study that the "air-wet technique" can be used to propagate Rhododendron species. This method also has the advantage of "true-to-type" propagation of elite or specific clones for mass multiplication required for reforestation programs. The study also revealed that the pot rooted cuttings grew well and can then be treated like other planting stocks in the nursery.

Acknowledgements
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. Mohan Kumar Thapa and Sunil are thanked for their assistance.

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Dr. K. K. Singh (Corresponding author) has an MSc in botany and PhD in plant physiology and biotechnology. He is currently working as a scientist in GB Pant Institute of Himalayan Environment and Development, Sikkim Unit. His research interests include in vitro mass multiplication of Rhododendron spp. and ex situ conservation of sensitive species.