VEGETATIVE PROPAGATION

VEGETATIVE PROPAGATION CHAPTER 7 7.1. INTRODUCTION A species perpetuates within the ecosystem by sexual and / or asexual reproduction. The former involves the fusion of male and female gametes, and the production of seeds, while in the latter a vegetative part of the plant, e.g., shoots, roots, buds or leaf, regenerates a new root system, a new shoot system or both, by mitotic divisions of cells. In vegetative propagation by stem, cuttings can be taken from shoots of the plants with terminal or lateral buds, which are capable of developing adventitious roots which finally emerge out to a complete plant (Hartmann et al., 1997). Thus in vegetative reproduction, the plants bear the same genetic traits from generation to generation. Such populations are called clones. Propagation through vegetative means assumes importance when desirable biotypes need to be multiplied in a short span of time. Horticulturists and foresters have been interested in rooting of stem cutting because; it is essential for largescale multiplication and domestication of many important plants. Most ornamentals, green house crops and numerous fruit and forest tree crops are propagated through vegetative propagation (stem cuttings). In addition, propagation by cutting is the only means of preserving unique characteristics of some plants. During recent past the stem cutting is extensively practiced by means of vegetative propagation in plants. However, the rooting success of the cuttings is dependent on various factors such as cutting size and diameter, rooting medium, hormonal concentration as well as bio-physical and environmental factors (Wilson, 1993). In order to meet the demand of development of adequate Hippophae rhamnoides (Seabuckthorn) resources with a regulated ratio of male and female plants, propagation from cutting needs to be preferred. Plants raised from the stem or branch cuttings maintain the genetic properties of the mother plants and female plants can bear fruit faster than the plants raised through seeds. Seabuckthorn is a dioecious, wind-pollinated plant. 92

The seedlings propagated by seeds cannot maintain the fine biological characteristics and economic properties that are genetically identical to the selected mother plants. Among the seedlings, usually there are more males then females, and it is difficult to distinguish males from females before flowering and fruiting. Propagation through cutting can produce scion-rooted seedlings with the same genetic properties as those of their mother plants within a short time, and can bear fruit 1-2 years earlier than the seedlings. This is an important tool for raising large number of planting materials in a short duration for undertaking rehabilitation/afforestation activities. Considering all these factors the present study was carried out to understand the vegetative propagation of the male and female plants of the target species with reference to rooting response and other parameters under different rooting media and hormonal treatments. 7.2. MATERIALS AND METHODS 7.2.1. Collection of stem cuttings The male and female cuttings were collected from all the five valleys (Niti, Mana, Bhyundar, Gangotri and Yamunotri) during the first week of March, 2005. Healthy and mature trees/shrubs were identified and selected for collecting stem cuttings. Both male and female plants were taken for the vegetative propagation experiments. To distinguish male and female plants in the nature were marked with two colours (Red for male and Green for female) during the fruiting period for proper identification. Branch cuttings of 15-20 cm size and 0.8 to 2.0 cm diameter, cleared for all leaves were collected and immediately kept in plastic film bags to prevent them from wind drying and withering. 7.2.2. Experimental setup An experiment on vegetative propagation of Hippophae rhamnoides (Seabuckthorn) was carried out under natural environmental conditions at an altitude of 2600 m amsl in village Tolma, located in Niti valley of Nanda Devi Biosphere Reserve (NDBR). The mean minimum and maximum aerial temperatures recorded during the study period were 28.05 0 C and 0.93 0 C, respectively (Chapter 5, Fig. 5.2). One set of male and female cuttings were kept as control and dipped in distilled water, the other were dipped in different concentration of Indole 3-acetic acid (IAA 25,,, 300, 0 and 0 93

ppm), Indole 3-butyric acid (IBA 25,,, 300, 0 and 0 ppm), and Naphthalene acetic acid (NAA 25,,, 300, 0 and 0 ppm). Each treatment has three replicates. The basal part of cuttings were placed at 2-3cm depth in the solution of growth hormone at room temperature for 24 hours, then properly washed with water and all the cuttings were planted in polythene bags containing pure soil and sand at the ratio 1:1 (soil: sand). After plantation, water was sprayed as per the requirements. Observations on stem sprouting and rooting were made fortnightly. The final rooting was recorded after a lapse of 90 days. However, the male and female cuttings were again uprooted out after 6 months of planting for estimating the root system. 7.3. RESULTS 7.3.1. Effect of Plant Growth Hormones on rooting The effect of Plant Growth Regulators (Hormones) viz., IAA, IBA and NAA (25,,, 300, 0 and 0 ppm) on rooting percentage of stem cuttings of H. rhamnoides is presented in Fig.7.1-7.6. The rooting ability of stem cuttings was observed after 3 months (90 days) of planting. The various treatments of PGRs (Hormones) with their concentrations (25,,, 300, 0 and 0 ppm) showed high significant differences (p<0.05) in both male and female cuttings of this plant species. The study indicated that the hormonal concentrations are the important factor for enhancing and stimulating the rooting percentage of stem cuttings. 7.3.1. A. Effect of IAA on male and female cuttings The results of rooting percentage for male and female cuttings of H. rhamnoides were significantly affected by various concentrations of IAA. Different concentrations viz., 25,,, 300, 0, 0 ppm showed different results for male and female cuttings. The best and maximum rooting percentage of male and female cuttings collected from different valleys was recorded under ppm of IAA. Amongst all five valleys, the Mana valley exhibited maximum rooting in soil as well as in mixture of soil and sand (1: 1). The male and female cuttings of Mana valley performed maximum rooting percentage with highest for female in soil (73.33±3.33 %) and in composite media (83.33±33 %). On the other hand the results of IAA 0 ppm showed the poor rooting performance for 94

male as well as for female cuttings. The minimum 6.67±3.33 % rooting in male cuttings were recorded in Bhyundar, Niti, Gangotri and Yamunotri valleys for soil as well as under composite media. The rooting percentage was also found very poor in female cuttings with least value (10.00±5.77 %) in soil for Gangotri valley and slightly higher (13.33±3.33 %) in composite media for Yamunotri valley. 7.3.1. B. Effect of IBA on male and female cuttings The percent of rooting in male and female cuttings collected from different valleys was significantly (p<0.05) affected by ppm of IBA. The maximum 70.00±5.77 % rooting in soil was recorded for the male cuttings of Mana valley whereas female cuttings of same valley showed highest (80.00±5.77 %) rooting percentage under soil media. The cuttings, which were planted in the composite media, showed increased rooting percentage. Among the valleys, the maximum rooting percentage for male and female cuttings was observed for Mana and Niti valleys, respectively (Table 7.1, 7.2). The higher concentration of IBA showed poor rooting response with minimum rooting was recorded in 0 ppm of IBA (Table 7.1, 7.2). Gangotri valley showed the minimum rooting 3.33±3.33 % for male and 13.33±8.82 % for female in soil whereas under composite media it showed 13.33±3.33 % of rooting for male and female cuttings of same valley. 7.3.1. C. Effect of NAA on male and female cuttings The lower concentrations of NAA resulted maximum rooting percentage whereas, its higher concentration showed low rooting percentage (Table 7.1, 7.2). NAA ppm was found significant (p<0.05) and has improved rooting percentages of male and female cuttings of different valleys. In soil condition the maximum rooting 60.00±5.77 % for male and 80.00±577 % for female was observed for the cuttings of Mana valley (Table 7.1). However, in composite rooting media (soil: sand) the rooting was higher 70.00±10.00 % for male and 83.33±3.33 % for female cuttings of Mana valley (Table 7.2) as compared to pure soil media. Gangotri valley showed the minimum rooting 10.00±5.77 % for male and 3.33±3.33 % for female under soil media while, 10.00±5.77 % and 13.33±8.82 % was recorded for male and female, respectively in composite rooting media. 95

A B C D E F Plate 7.1. Showing details about vegetative propagation: (A) Experimental nursery of stem cuttings, (B) early sprouted cuttings, (C) established plants in polybags, (D) profused rooting after 6 months of cutting planted in nursery, (E) rooted stem cuttings, (F) plantation of well developed cuttings 96

Table 7.1. Effect of Plant growth hormones on rooting of male and female cuttings of H. rhamnoides, collected from five valleys under soil media. Valley Gangotri Yamunotri Bhyundar Mana Niti Significant, p<0.05, Conc. (ppm) Control 25 300 0 0 Control 25 300 0 0 Control 25 300 0 0 Control 25 300 0 0 Control 25 300 0 0 Hormone IAA IBA NAA 26.67±3.3 40.00±5.8 - - - - 43.33±3.3.00±5.8.00±5.8 53.33±6.7 36.67±8.2 26.67±12.0 56.67±3.3 60.00±5.8.00±5.8 76.67±3.3.00±5.8.00±5.8 30.00±5.8 36.67±8.8 23.33±6.7 33.33±3.3 23.33±8.8 30.00±5.8 30.00±11.5 13.33±8.8 20.00±5.8 20.00±5.8 30.00±5.8 10.00±5.8 20.00±11.5 23.33±8.8 13.33±5.8 26.67±5.8 13.33±5.8 13.33±5.8 10.00±5.8 10.00±5.8 3.33±3.3 13.33±8.8 10.00±5.8 3.33±3.3 30.00±10.0 43.33±12.0 - - - - 30.00±5.78 30.00±5.8 23.33±8.8 36.67±3.3 16.67±3.3 23.33±3.3 40.00±5.8 56.67±5.8 33.33±8.8 33.33±3.3 43.33±3.3 43.33±3.3 10.00±5.8 40.00±5.8 20.00±5.8 10.00±5.8 16.67±3.3 36.67±6.7 20.00±5.8 26.67±6.7 20.00±5.8 33.33±3.3 20.00±0.0 23.33±8.8 13.33±8.8 10.00±5.8 13.33±3.3 30.00±5.8 16.67±3.3 23.33±3.3 13.33±3.3 13.33±3.3 20.00±10.0 13.33±8.8 13.33±3.3 23.33±8.8 30.00±5.8 40.00±5.8 - - - - 23.33±3.3 53.33±6.7 30.00±5.8 53.33±3.3 30.00±5.8 56.67±8.8 33.33±3.3 70.00±6.7 40.00±5.8.00±3.3 53.33±5.8 70.00±8.8 16.67±3.3 33.33±6.7 36.67±5.8 53.33±3.3 26.67±5.8 46.67±8.8 16.67±6.7 30.00±5.8 10.00±5.8 26.67±8.8 16.67±3.3 20.00±5.8 10.00±0.0 30.00±5.8 20.00±5.8 40.00±5.8 13.33±3.3 23.33±8.8 6.67±3.3 13.33±3.3 13.33±3.3 20.00±5.8 13.33±3.3 30.00±11.5 23.33±3.3 40.00±5.8 - - - - 56.67±3.3 66.67±3.3 60.00±5.8 73.33±3.3 63.33±3.3 80.00±0.0 63.33±3.3 73.33±3.3 70.00±5.8 80.00±5.8 60.00±5.8 80.00±58 46.67±6.7 63.33±8.8 56.67±8.8 66.67±8.8 46.67±6.7 53.33±8.8 53.33±8.8 46.67±3.3 40.00±5.8 46.67±14.5 40.00±5.8 40.00±5.8 23.33±3.3 26.67±8.8 16.67±3.3 33.33±6.7 20.00±5.8 23.33±3.3 23.33±3.3 26.67±8.8 20.00±5.8 23.33±8.8 13.33±3.3 13.33±3.3 33.33±6.7 43.33±3.3 - - - - 43.33±3.3 66.67±3.3 46.67±8.8 60.00±5.8 43.33±8.8 73.33±3.3 43.33±8.8 66.67±3.3 53.33±3.3 73.33±8.8 40.00±5.8 56.67±8.8 33.33±3.3 46.67±3.3 26.67±6.7 33.33±8.8 30.00±5.8.00±5.8 26.67±3.3 33.33±3.3 23.33±3.3 30.00±0.0 16.67±3.3 33.33±8.8 20.00±5.8 26.67±3.3 13.33±8.8 33.33±3.3 16.67±8.8 20.00±0.0 6.67±3.3 13.33±3.3 3.33±3.3 20.00±5.8 10.00±5.8 20.00±0.0 97

Table 7.2. Effect of Plant growth hormones on rooting of male and female cuttings of H. rhamnoides, collected from five valleys under composite media (soil: sand). Valley Gangotri Yamunotri Bhyundar Mana Niti Significant, p<0.05, Conc. (ppm) Control 25 300 0 0 Control 25 300 0 0 Control 25 300 0 0 Control 25 300 0 0 Control 25 300 0 0 Hormone IAA IBA NAA 43.33±3.3 43.33±6.7 - - - - 53.33±3.3 66.67±3.3 43.33±6.7 60.00±5.8 36.67±8.8 46.67±3.3 63.33±6.7 70.00±5.8 53.33±3.3 80.00±5.8 46.67±3.3.00±5.8 33.33±3.3 46.67±8.8 23.33±12.0 33.33±6.7 40.00±5.8 33.33±3.3 23.33±6.7 43.33±8.8 20.00±5.8 6.67±3.3 26.67±8.8 36.67±12.0 16.67±6.7 30.00±8.8 16.67±5.8 30.00±3.3 26.67±8.8 26.67±12.0 6.67±3.3 20.00±5.8 13.33±3.3 13.33±3.3 10.00±5.8 13.33±8.8 30.00±11.5 33.33±3.3 - - - - 36.67±3.3.00±0.0 23.33±3.3 53.33±8.8 40.00±5.8 53.33±3.3 40.00±3.3 40.00±0.0 30.00±3.3 60.00±8.8 36.67±5.8 53.33±3.3 16.67±6.7.00±5.8 16.67±3.3 36.67±8.8 20.00±5.8 26.67±14.5 10.00±0.0 10.00±0.0 23.33±6.8 26.67±8.8 20.00±5.8 36.67±8.8 26.67±3.3 33.33±3.3 26.67±3.3 23.33±14.5 13.33±8.8 20.00±5.8 6.67±3.3 13.33±3.3 23.33±8.8 30.00±5.8 23.33±3.3 30.00±15.3 36.67±8.8.00±5.8 - - - - 40.00±5.8 56.67±6.7 30.00±5.8 66.67±8.8 40.00±5.8 60.00±5.8 53.33±5.8 66.67±6.7 53.33±5.8 80.00±8.8 66.67±58 70.00±5.8 43.33±5.8.00±6.7 43.33±5.8 66.67±8.8 43.33±5.8 53.33±5.8 20.00±5.8 30.00±11.5 16.67±6.8 46.67±3.3 13.33±3.3 26.67±3.3 20.00±5.8 40.00±11.5 13.33±8.8 33.33±6.7 23.33±3.3 33.33±3.3 6.67±6.7 30.00±5.8 13.33±6.7 33.33±3.3 10.00±5.8 23.33±3.3 33.33±8.8.00±5.8 - - - - 70.00±5.8 76.67±6.7 66.67±8.8 73.33±6.7 76.67±3.3 76.67±8.8 66.67±8.8 83.33±3.3 76.67±6.7 80.00±5.8 70.00±10.0 83.33±3.3 46.67±6.7 56.67±12.0 66.67±6.7 63.33±8.8 56.67±6.7 56.67±8.8 46.67±6.7 43.33±8.8 56.67±3.3 53.33±12.0 40.00±11.5 40.00±10.0 33.33±8.8 33.33±14.5 16.67±6.7 23.33±8.8 26.67±8.8 26.67±6.7 23.33±8.8 33.33±12.0 20.00±10.0 23.33±13.3 13.33±3.3 16.67±3.3 36.67±3.3 53.33±3.3 - - - -.00±5.8 76.67±3.3 60.00±0.0 66.67±8.8 60.00±5.8 76.67±6.7 53.33±3.3 80.00±5.8 53.33±8.8 83.33±3.3 46.67±3.33 80.00±58 26.67±6.7 56.67±6.7 26.67±3.3 46.67±6.7 40.00±11.5 56.67±6.7 26.67±6.7 43.33±3.3 16.67±3.3 36.67±3.3 23.33±8.8 56.67±8.2 10.00±5.8 30.00±10.0 23.33±3.3 43.33±6.7 33.33±3.3.00±0.0 23.33±8.8 23.33±8.8 16.67±8.8 36.67±8.8 16.67±6.7 10.00±5.8 98

7.3.2. Effect of Hormones and rooting media on other parameters of root The other parameters of root i.e. number of root/shoot, root length and root diameter were also considered to see the responses of different hormones and rooting media on male and female cuttings of Hippophae rhamnoides. 7.3.2.(i) Number of roots / shoot: The results of various hormonal concentrations showed significant differences (p<0.05) in the number of roots per shoot for male and female cuttings in the cuttings of all the five valleys (Table 7.3, 7.4). However, the results of cuttings of all valleys were approximately more or less same but the application of different hormones and their concentrations showed useful effects over the number of roots per shoot. The maximum number of roots per shoot (soaked in NAA 25 ppm) was recorded 3.3±0.3 for male and 4.3±0.9 for female cuttings of Bhyundar valley with soil condition whereas, it was found very low 1.0±0.0 for male and 1.7±0.7 for female cuttings (soaked in IAA 0 ppm) of Gangotri valley (Table 7.3). On the other hand the results of composite rooting media showed maximum 3.3±0.3 for male cuttings (soaked in IBA 25 ppm) of Yamunotri valley and 5.3±0.3 for female cuttings of Bhyundar with 25 ppm of NAA. The cuttings of Niti valley treated with IAA 0 ppm exhibited 1.3±0.3 and 2.3±0.7 roots per shoot for male and female cuttings, respectively (Table 7.4). 7.3.2. (ii) Root Length: The results of root length were also affected with the application of different concentrations of hormones in all valleys. The cuttings of Mana valley which were soaked into IBA ppm performed the maximum root length 3.8±0.7 cm for male and 5.1±0.8 cm for female in soil media while it was measured 0.5±0.1 cm for male cuttings of Bhyundar valley and 0.9±0.3 cm for female cuttings of Niti valley soil media (Table 7.5). On the other hand under composite media the length of root was found maximum 5.7±0.6 cm for male and 6.2±1.2 cm for female cuttings of Niti valley when treated with IBA ppm. IBA 0 ppm showed minimum length of root 0.7±0.3 99

cm for male cuttings of Yamunotri valley and 1.0±0.3 cm for female of Niti valley under the composite rooting media (Table 7.6). 7.3.2.(iii) Root Diameter: The observation in relation to root diameter did not show much variation and also did not show significant differences (p>0.05) between male and female cuttings of H. rhamnoides while, some of the concentrations showed significant differences (p<0.05) for increasing the diameter of root. The maximum diameter of male cuttings (2.2±0.2 mm) was recorded in NAA 25 ppm for Mana valley under soil condition while 2.3±0.2 mm diameter was recorded for female cuttings of Niti valley in soil media with same concentration (Table 7.7). The cuttings treated with IAA 0 and IBA 0 exhibited very low diameter for male cuttings while IAA 0 ppm exhibited 1.5±0.2 mm diameter for female cuttings of Mana and Niti valleys. On the other hand the composite media showed maximum 2.3±0.1 mm diameter under 25 ppm of NAA for the female cuttings (Niti valley) wheras, it was recorded 2.1±0.1 mm for male cuttings of Bhyundar valley (Table 7.8). The least value of root diameter (1.4±0.1 mm) was observed under 0 ppm of IBA for male cuttings of Gangotri valley whereas, it was 1.6±0.3 mm under 0 ppm of IBA for the female cuttings of Gangotri valley under composite media of soil and sand (Table 7.8). Table 7.3. Effect of plant growth hormones on the number of roots/shoot of male and female cuttings of H. rhamnoides under pure soil condition. Treatment M F M F M F M F M F IAA 25 2.7±0.9 3.7±0.3 2.3±0.7 4.0±06 2.7±0.3 3.7±0.3 2.7±0.3 3.7±0.3 2.3±0.3 3.0±0.6 2.3±0.7 3.3±0.3 2.3±0.7 3.7±0.3 2.3±0.3 2.7±0.3 2.3±0.3 2.3±0.7 2.0±0.6 2.0±0.6 2.0±0.0 3.7±0.7 1.7±0.3 3.0±1.2 2.3±0.3 3.0±0.6 2.3±0.3 3.0±0.6 2.3±0.9 3.0±0.6 300 2.7±0.9 3.3±0.9 3.0±0.6 3.7±0.9 2.7±0.9 3.7±0.6 2.7±0.9 3.3±0.9 2.0±1.0 3.3±0.9 0 2.0±0.6 3.0±1.2 1.7±0.7 2.3±0.7 1.7±0.7 2.0±0.9 1.7±0.7 2.0±0.6 1.3±0.3 1.7±0.7 0 1.0±0.0 1.7±0.7 2.0±0.6 2.0±0.6 1.7±0.7 2.7±0.3 2.0±0.6 2.3±0.3 1.3±0.3 2.0±0.6 IBA 25 2.7±0.9 3.3±0.3 3.0±0.6 3.7±0.7 3.0±0.6 4.3±0.9 2.7±0.3 4.3±0.3 3.3±0.3 4.0±0.6 2.3±0.7 3.0±1.2 1.7±0.7 3.0±0.9 2.7±0.3 3.0±0.6 2.0±0.6 3.3±0.3 2.7±0.3 2.7±0.3 2.3±0.7 3.0±0.0 2.3±0.7 2.7±0.6 1.7±0.3 3.3±0.3 2.7±0.3 4.0±0.6 3.0±0.6 4.0±0.6 300 1.7±0.7 4.0±0.6 2.3±0.7 3.0±0.6 2.7±0.9 3.0±0.6 3.0±0.6 3.3±0.3 3.0±0.6 3.3±0.3 0 2.7±0.3 3.0±0.0 2.7±0.9 3.3±0.7 2.7±0.9 2.7±0.3 2.0±0.6 3.0±0.0 2.0±0.6 2.0±0.6 0 1.7±0.7 2.0±0.6 1.7±0.7 2.7±0.9 1.7±0.7 2.3±0.7 2.0±0.6 2.3±0.3 2.0±0.6 2.7±0.9 NAA 25 2.7±0.9 4.0±0.6 2.3±0.7 4.0±0.6 3.3±0.3 4.3±0.9 2.7±0.7 4.0±0.6 2.7±0.7 3.7±0.9 3.0±0.6 4.0±0.6 2.3±0.9 3.0±0.6 2.7±0.7 4.0±0.6 2.7±0.3 3.3±0.3 2.3±0.7 3.0±0.6 2.0±0.6 3.3±0.3 2.0±0.6 3.0±0.6 2.0±1.0 3.0±0.6 2.3±0.3 3.3±0.9 2.0±0.6 3.7±0.9 300 1.7±0.7 3.3±1.2 2.0±0.6 2.7±0.9 1.7±0.7 3.0±0.6 2.3±0.3 3.3±0.3 2.0±0.6 3.3±0.3 0 1.7±0.7 3.0±0.6 1.7±0.7 2.3±0.7 2.0±0.6 2.7±0.3 2.0±0.6 2.7±0.3 1.7±0.7 2.3±0.7 0 1.7±0.7 2.0±0.6 2.7±0.9 2.0±0.6 1.7±0.7 2.0±0.6 1.7±0.7 3.0±0.6 1.7±0.7 2.3±0.7 Control 2.3±0.3 3.7±0.3 2.7±0.3 3.0±0.6 2.3±0.3 2.7±0.9 2.7±0.3 3.0±0.6 2.0±0.6 3.0±0.6 P<0.05; M=, F=

Table 7.4. Effect of plant growth hormones on the number of roots/shoot of male and female cuttings of H. rhamnoides under soil:sand condition. Treatment M F M F M F M F M F IAA 25 3.3±0.7 3.7±0.9 3.3±0.7 4.3±0.3 3.3±0.3 4.7±0.3 3.0±0.6 4.7±0.3 2.0±0.3 4.0±0.6 2.7±0.3 4.3±0.3 3.0±0.6 4.0±0.6 2.7±0.3 3.7±0.3 2.3±0.7 3.3±0.3 2.0±0.6 3.0±0.6 3.3±0.3 2.3±1.3 2.0±0.6 3.7±0.7 3.0±0.6 3.3±0.3 3.0±0.6 3.0±0.6 2.7±0.9 2.0±0.6 300 2.0±0.6 3.3±1.3 3.0±0.6 4.0±0.6 3.0±0.6 4.0±0.6 3.0±0.6 3.3±0.9 2.0±1.0 3.7±0.9 0 2.3±0.7 2.3±1.3 2.3±0.3 3.7±0.9 2.3±0.7 3.3±0.3 2.0±0.6 3.0±0.6 1.3±0.3 2.3±0.7 0 2.0±0.6 3.3±0.3 1.7±0.7 2.7±0.9 2.3±0.7 2.7±0.9 2.0±0.6 2.3±0.3 2.0±0.3 2.7±0.6 IBA 25 3.3±0.9 4.7±0.9 3.3±0.3 5.0±0.0 3.7±0.3 4.7±0.9 3.0±0.6 4.0±0.6 3.0±0.3 4.7±0.6 3.3±0.9 3.3±0.3 3.7±0.3 3.7±0.3 3.3±0.3 4.0±0.6 3.3±0.3 4.0±0.6 3.3±0.3 3.7±0.3 2.7±0.3 3.3±0.3 3.0±0.6 3.3±0.3 3.0±0.6 4.0±0.6 3.7±0.3 4.0±0.6 3.3±0.6 4.3±0.6 300 3.0±0.6 3.0±1.2 2.0±0.6 3.3±0.9 2.3±0.7 3.7±1.2 2.3±0.3 4.0±0.6 2.0±0.6 4.0±0.3 0 2.0±0.6 2.7±0.3 2.7±1.2 3.0±0.6 3.0±0.6 3.0±0.6 2.7±0.3 3.0±0.6 2.3±0.6 3.3±0.6 0 1.7±0.7 2.3±0.7 2.7±0.3 3.0±0.0 2.0±0.6 3.0±0.6 2.0±0.6 3.0±0.6 2.7±0.6 3.0±0.9 NAA 25 2.7±0.3 4.0±0.0 2.7±0.3 3.7±0.9 2.7±0.3 5.3±0.3 3.0±0.6 4.7±0.3 3.0±0.7 4.3±0.9 2.0±0.6 3.7±0.7 2.7±0.3 3.7±0.7 3.0±0.6 3.3±0.9 3.0±0.0 3.7±0.3 2.7±0.7 3.7±0.6 2.7±1.2 3.0±0.6 2.7±0.9 3.0±0.0 3.3±0.9 2.7±0.9 3.0±0.6 3.3±0.9 3.0±0.6 3.3±0.9 300 2.3±0.7 2.7±0.9 2.3±0.3 3.3±0.9 2.0±0.6 3.3±0.9 2.7±0.3 3.7±0.3 2.3±0.6 3.7±0.3 0 1.7±0.7 2.3±0.9 1.7±0.7 3.0±0.6 2.3±0.7 3.0±0.6 2.0±0.6 3.3±0.3 2.0±0.7 3.0±0.7 0 2.0±0.6 2.3±0.7 1.7±0.7 3.0±1.2 1.7±0.7 2.3±0.7 2.7±0.9 2.7±0.9 2.7±0.7 2.7±0.7 Control 3.0±0.6 4.0±0.6 2.3±0.9 4.0±0.6 2.7±0.7 3.0±0.0 3.0±0.0 2.7±0.3 2.3±0.7 2.3±0.7 P<0.05; M=, F= Table 7.5. Effect of plant growth hormones on the root length (cm) of male and female cuttings of H. rhamnoides under soil condition. Treatment M F M F M F M F M F IAA 25 3.1±0.3 4.2±0.4 3.1±0.1 4.5±0.6 2.9±0.2 4.2±0.7 3.9±0.1 4.6±0.5 3.3±0.2 4.5±0.5 2.8±0.3 4.2±0.8 2.8±0.3 3.8±0.8 3.5±0.5 4.0±0.5 3.6±0.6 4.7±0.6 3.8±0.6 4.7±0.5 1.9±0.5 4.1±0.4 2.0±0.4 4.0±0.4 2.5±0.1 3.8±0.5 2.3±0.4 4.4±0.3 2.6±0.4 3.9±0.3 300 2.3±0.5 2.1±0.5 1.9±0.4 2.1±0.6 1.9±0.1 2.5±0.1 2.0±0.3 2.9±0.5 1.7±0.2 2.6±0.5 0 0.9±0.4 1.8±0.3 1.1±0.3 1.9±0.1 0.7±0.2 1.3±0.3 1.0±0.2 1.2±0.3 0.7±0.2 1.2±0.2 0 1.0±0.3 1.5±0.4 0.8±0.1 1.3±0.1 0.5±0.1 1.4±0.2 1.0±0.1 1.3±0.3 0.7±0.2 0.9±0.3 IBA 25 2.9±0.3 3.6±0.4 3.1±0.6 3.3±0.6 2.8±0.2 3.2±0.4 3.6±0.5 4.4±0.4 3.6±0.6 4.1±0.4 2.5±0.4 3.5±0.4 2.4±0.2 3.6±0.4 2.5±0.1 3.9±0.2 3.8±0.7 5.1±0.8 3.4±0.7 4.3±0.2 2.5±0.2 3.5±0.4 2.5±0.2 3.4±0.4 2.5±0.1 3.8±0.4 3.0±0.3 4.8±0.2 2.6±0.3 4.0±0.5 300 2.4±0.5 3.4±0.6 2.2±0.2 3.4±0.5 2.3±0.1 3.7±0.4 2.8±0.5 4.0±0.2 2.3±0.4 3.6±0.4 0 2.1±0.2 2.2±0.6 1.4±0.1 1.7±0.2 1.4±0.2 1.8±0.2 1.6±0.3 1.6±0.5 1.3±0.3 1.7±0.1 0 1.0±0.4 1.3±0.3 1.1±0.4 1.1±0.3 1.3±0.3 1.1±0.3 1.0±0.2 1.2±0.1 0.7±0.2 1.0±0.1 NAA 25 3.1±0.7 3.3±0.5 3.3±0.5 3.2±0.3 3.2±0.6 3.4±0.2 4.0±0.4 4.1±0.5 3.6±0.3 3.7±0.3 3.0±0.3 3.3±0.5 2.7±0.5 3.3±0.8 2.6±0.6 3.5±0.6 3.1±0.3 4.3±0.1 3.0±0.3 3.4±0.4 2.0±0.3 3.1±0.3 1.6±0.1 2.8±0.4 1.6±0.2 2.8±0.3 2.3±0.6 3.1±0.2 2.3±0.5 3.0±0.5 300 1.7±0.4 2.6±1.0 1.6±0.3 3.0±0.2 1.6±0.4 2.6±0.2 2.1±0.2 2.8±0.2 1.6±0.3 2.7±0.7 0 1.5±0.4 1.4±0.5 1.2±0.7 1.5±0.3 1.2±0.5 1.6±0.4 1.7±0.4 1.9±0.2 1.4±0.4 1.5±0.3 0 1.5±0.3 1.5±0.7 1.1±0.1 1.4±0.4 1.1±0.3 1.4±0.1 0.7±0.2 1.1±0.2 1.0±0.5 1.7±0.3 Control 2.2±0.2 3.3±0.4 2.2±0.3 3.0±0.6 2.4±0.5 3.5±0.5 2.4±0.7 3.9±0.2 2.6±0.3 3.3±0.3 P<0.05; M=, F= 101

Table 7.6. Effect of plant growth hormones on the root length (cm) of male and female cuttings of H. rhamnoides under soil:sand condition. Treatment M F M F M F M F M F IAA 25 4.0±0.4 4.6±0.5 4.3±o.4 4.7±0.8 4.8±0.3 5.2±0.5 5.1±o.3 6.0±0.8 5.1±0.2 5.8±0.6 3.9±0.2 5.1±0.2 4.0±0.3 4.8±0.3 4.2±0.6 5.2±0.3 4.4±0.6 5.5±0.3 4.6±0.3 5.5±0.3 2.4±0.1 4.2±0.3 2.6±0.2 4.7±0.2 3.0±0.4 4.6±0.2 3.0±0.1 4.9±0.4 2.4±0.1 4.4±0.4 300 2.8±0.5 3.0±0.4 2.1±0.7 3.7±0.1 2.0±0.2 2.6±0.5 2.3±0.4 3.2±0.4 2.1±0.5 2.9±0.4 0 1.1±0.3 1.7±0.4 1.1±0.3 2.1±0.1 1.0±0.3 1.4±0.6 1.5±0.2 1.9±0.4 1.0±0.1 1.6±0.3 0 1.2±0.2 2.0±0.2 1.0±0.4 1.5±0.1 1.0±0.3 1.7±0.2 1.2±0.4 2.0±0.2 0.9±0.2 1.2±0.4 IBA 25 3.5±0.4 4.2±0.2 3.7±0.4 4.6±0.2 4.0±0.3 4.6±0.4 4.7±0.4 5.1±0.5 4.3±0.5 5.1±0.5 3.6±0.1 4.1±0.1 3.6±0.2 4.5±0.5 4.3±0.4 4.6±0.5 5.7±0.5 5.6±1.3 5.7±0.6 6.2±1.2 2.8±0.3 3.6±0.6 2.7±0.3 3.6±0.3 2.9±0.3 4.5±0.6 3.9±0.2 5.0±0.4 3.3±0.3 4.8±0.2 300 2.9±0.6 3.7±0.6 2.7±0.2 4.1±0.5 3.0±0.4 4.2±0.5 2.9±0.9 3.8±0.7 2.9±0.4 3.8±0.5 0 1.9±0.5 3.0±0.2 1.5±0.2 2.5±0.3 1.9±0.5 2.2±0.7 1.8±0.3 2.0±0.2 1.5±0.2 2.0±0.3 0 1.3±0.4 1.9±0.3 0.7±0.3 1.2±0.3 0.9±0.4 1.3±0.2 1.3±0.5 1.5±0.1 1.4±0.3 1.0±0.3 NAA 25 3.8±0.6 4.7±0.5 3.5±0.4 4.4±0.8 3.7±0.4 5.3±0.5 4.6±0.4 5.6±0.2 4.0±0.5 5.3±0.6 3.0±0.3 3.4±0.7 3.1±0.2 3.7±0.4 3.5±0.3 4.6±0.4 3.6±0.7 4.1±0.4 3.5±0.4 4.8±0.2 2.1±0.4 3.6±0.4 2.3±0.5 3.8±0.7 2.5±0.5 3.8±0.9 3.0±0.9 5.2±1.0 3.0±0.6 4.1±0.8 300 2.8±0.4 3.2±0.8 2.7±0.3 3.2±0.2 2.8±0.4 3.6±0.6 2.8±0.2 3.7±0.5 2.9±0.3 3.4±0.5 0 2.2±0.4 1.6±0.4 1.5±0.7 1.4±0.4 1.6±0.2 1.3±0.2 1.7±0.5 1.8±0.5 1.2±0.4 1.8±0.3 0 1.3±0.6 1.9±0.1 1.3±0.5 1.2±0.1 1.3±0.1 1.6±0.4 1.5±0.1 1.9±0.4 1.0±0.3 1.5±0.5 Control 2.1±0.4 2.9±0.7 2.5±0.3 3.5±0.5 2.8±0.4 3.8±0.4 2.4±0.4 3.9±0.7 2.6±0.4 3.8±0.7 P<0.05; M=, F= Table 7.7. Effect of plant growth hormones on the root diameter (mm) of male and female cuttings of H. rhamnoides under soil condition. Treatment M F M F M F M F M F IAA 25 1.6±0.1 2.0±0.2 1.9±0.1 1.9±0.1 1.5±0.1 1.7±0.2 1.7±0.3 1.9±0.2 1.6±0.2 1.6±0.2 1.9±0.1 1.8±0.2 1.7±0.1 2.0±0.1 1.6±0.2 1.5±0.2 1.7±0.1 1.9±0.1 1.6±0.1 1.5±0.2 1.8±0.1 1.9±0.2 1.9±0.1 1.9±0.1 1.5±0.3 1.9±0.2 1.4±0.2 1.8±0.2 1.1±0.1 1.8±0.2 300 1.8±0.2 1.9±0.3 1.8±0.3 1.8±0.2 1.6±0.3 1.7±0.2 1.3±0.3 1.7±0.3 1.1±0.1 1.6±0.2 0 1.7±0.2 1.7±0.2 1.6±0.3 1.9±0.1 1.2±0.3 1.8±0.1 1.5±0.3 1.9±0.1 1.1±0.2 1.7±0.2 0 1.7±0.2 1.7±0.3 1.6±0.2 1.6±0.2 1.9±0.1 1.6±0.2 1.6±0.4 1.5±0.2 1.6±0.3 1.5±0.2 IBA 25 2.1±0.1 2.1±0.1 1.9±0.1 1.8±0.2 1.9±0.1 2.0±0.2 2.0±0.1 2.0±0.2 1.9±0.1 1.9±0.1 1.7±0.3 1.9±0.1 1.7±0.2 2.0±0.1 1.8±0.3 1.8±0.1 1.9±0.1 2.0±0.1 1.8±0.1 1.8±0.1 1.4±0.2 2.0±0.1 1.7±0.1 2.0±0.1 1.8±0.2 2.1±0.4 1.7±0.2 2.0±0.2 1.6±0.2 2.1±0.1 300 1.4±0.2 1.7±0.3 1.6±0.2 1.9±0.2 1.3±0.3 2.0±0.2 1.6±0.3 2.2±0.2 1.3±0.3 1.9±0.1 0 1.2±0.2 1.8±0.3 1.3±0.3 1.7±0.3 1.4±0.1 1.6±0.1 1.6±0.2 1.8±0.3 1.1±0.2 1.8±0.3 0 1.6±0.1 2.1±0.1 1.7±0.3 2.0±0.3 1.7±0.3 1.8±0.3 1.8±0.1 2.0±0.1 1.7±0.4 2.1±0.3 NAA 25 1.8±0.2 2.1±0.2 1.8±0.3 2.2±0.2 2.0±0.2 2.2±0.1 2.2±0.2 2.2±0.1 2.0±0.2 2.3±0.2 1.9±0.2 2.1±0.2 1.8±0.2 1.9±0.1 1.8±0.2 2.1±0.2 2.1±0.1 2.0±0.2 1.9±0.1 2.2±0.2 1.5±0.2 2.0±0.2 1.7±0.2 1.9±0.2 1.7±0.2 1.9±0.1 1.6±0.2 1.9±0.2 1.7±0.2 1.9±0.2 300 1.5±0.2 1.9±0.1 1.9±0.1 2.0±0.2 1.5±0.2 1.9±0.1 1.5±0.2 2.1±0.2 1.4±0.2 2.1±0.1 0 1.5±0.0 2.0±0.2 1.6±0.2 1.9±0.3 1.4±p.3 1.7±0.2 1.7±0.1 1.8±0.2 1.5±0.3 1.8±0.2 0 1.7±0.2 2.0±0.2 1.7±0.2 2.0±0.2 1.8±0.1 2.1±0.1 1.8±0.0 1.9±0.2 1.8±0.1 2.0±0.1 Control 1.8±0.1 1.8±0.2 1.9±0.2 1.9±0.4 2.0±0.2 2.1±0.1 1.9±0.2 2.0±0.2 1.6±0.3 1.9±0.2 P<0.05; M=, F= 102

Table 7.8. Effect of plant growth hormones on the root diameter (mm) of male and female cuttings of H. rhamnoides under soil:sand condition. Treatment M F M F M F M F M F IAA 25 1.8±0.1 2.0±0.2 1.8±0.1 2.1±0.1 1.7±0.0 2.1±0.1 1.9±0.1 2.1±0.1 1.6±0.1 2.1±0.1 1.8±0.3 1.8±0.2 1.8±0.2 1.8±0.2 1.8±0.2 1.8±0.1 1.7±0.1 2.1±0.1 1.6±0.1 1.9±0.1 1.9±0.3 1.8±0.1 1.8±0.1 2.0±0.2 1.9±0.1 2.0±0.2 1.9±0.1 1.9±0.1 2.0±0.2 1.9±0.1 300 1.7±0.2 1.7±0.2 1.8±0.1 1.8±0.1 1.6±0.1 1.9±0.1 1.7±0.2 1.9±0.1 1.4±0.2 2.0±0.2 0 1.6±0.1 2.0±0.1 1.5±0.2 1.9±0.1 1.5±0.2 1.9±0.3 1.5±0.1 2.1±0.1 1.4±0.1 1.8±0.3 0 1.9±0.1 1.8±0.3 1.7±0.1 1.7±0.2 1.7±0.2 1.7±0.3 1.7±0.2 1.7±0.2 1.7±0.1 1.7±0.2 IBA 25 2.1±0.2 1.9±0.3 1.8±0.3 1.9±0.4 1.7±0.4 2.0±0.2 2.1±0.2 2.1±0.1 2.0±0.2 2.0±0.1 1.7±0.2 2.0±0.3 1.9±0.1 2.0±0.2 2.0±0.1 2.2±0.2 2.1±0.1 2.2±0.2 2.1±0.1 2.2±0.2 2.0±0.1 1.8±0.3 2.0±0.2 2.0±0.2 1.8±0.1 2.0±0.2 1.9±0.1 2.1±0.1 1.9±0.1 2.2±0.3 300 1.6±0.2 1.8±0.2 1.6±0.3 1.9±0.2 1.9±0.3 1.9±0.1 1.9±0.2 2.0±0.3 1.8±0.2 1.9±0.2 0 1.4±0.1 1.6±0.3 1.6±0.2 2.0±0.2 1.6±0.1 1.8±0.4 1.6±0.1 1.8±0.3 1.6±0.1 1.7±0.3 0 2.0±0.3 2.0±0.2 1.7±0.2 1.9±0.2 1.7±0.2 2.0±0.1 1.8±0.2 2.0±0.2 1.7±0.2 1.9±0.3 NAA 25 1.9±0.2 2.1±0.2 2.1±0.1 2.2±0.1 2.0±0.2 2.2±0.2 2.0±0.2 2.3±0.2 2.0±0.2 2.3±0.1 1.9±0.1 1.9±0.1 2.2±0.2 2.1±0.2 2.1±0.1 2.2±0.2 2.2±0.2 2.2±0.1 2.1±0.1 2.2±0.2 2.0±0.2 2.2±0.1 1.7±0.2 2.1±0.1 1.8±0.1 2.0±0.1 2.0±0.1 2.3±0.1 1.9±0.1 2.2±0.2 300 1.6±0.0 1.9±0.1 1.7±0.2 2.0±0.1 1.7±0.2 1.9±0.2 1.8±0.2 1.9±0.1 1.6±0.2 2.0±0.0 0 1.7±0.2 1.9±0.1 1.8±0.2 1.8±0.3 1.5±0.1 1.9±0.1 1.6±0.1 2.0±0.4 1.6±0.2 1.9±0.1 0 1.9±0.2 2.0±0.2 1.7±0.2 1.8±0.2 1.8±0.2 2.1±0.1 1.8±0.2 1.9±0.2 1.8±0.2 2.1±0.2 Control 1.9±0.1 1.9±0.2 1.9±0.2 2.2±0.2 2.0±0.2 1.7±0.4 1.9±0.1 2.0±0.1 1.9±0.1 1.8±0.3 P<0.05; M=, F= 7.4. DISCUSSION The results of these experiments highlighted that the Seabuckthorn (H. rhamnoides) stem cuttings of male and female plants could be propagated successfully. Optimum rooting percentage of cuttings was recorded, which implies that effective vegetative propagation of this species could be feasible. The natural habitat (original climatic conditions) at 2600m amsl, was selected and used for undertaking in-depth vegetative propagation experiments. Rooting response under control treatment clearly indicate the necessity of the different treatments of plant growth hormones to initiate the better rooting percentage. It was observed that the lower concentrations (25 and ppm) of various plant growth hormones provide better results of vegetative propagation. The study is also strengthened by the Garonovich (2003), who recommended treatment of cuttings with IBA ( mg/lit), IBA ( mg/lit) and NAA ( mg/lit) for 16 hours. On the other hand, the higher concentrations (, 300, 0 and 0 ppm) of hormones have shown poor rooting response over the male and female cuttings (Fig. 7.3-7.6). The results (Fig. 7.2) indicated that the IBA ppm increased the rooting percentage of male and female cuttings followed by NAA ppm and IAA ppm. The male and female cuttings collected from 103

Mana valley showed highest 70.00±5.77 % and 80.00±5.77 % of rooting in IBA ppm under soil media. Moreover, better responses with IBA were in conformity with the reports of its effectiveness as compared to several naturally occurring auxins in promotion of adventitious roots (Hartmann and Kester, 1983). Rooting media also plays a key role for improving the rooting results in the same cuttings under the same treatment (Leakey, 1990; Berhe and Negash, 1998). Under the composite rooting media of soil and sand (1: 1) the plant developed 76.67±6.67 % and 80.00±5.77 % of rooting for male and female cuttings treated with IBA ppm. The study is supported by the work carried out by Shuhua et al. (1989) as their work indicated that the sand being porous (higher oxygen), is an important medium to improve the rooting percentage of H. rhamnoides (Singh and Gupta, 2003). The results of rooting percentage obtained in soil media revealed that the mixture of soil and sand improved the rooting percentage up to 16.66 % (26.67±3.33 % to 43.33±3.33 %) under control treatment particularly for male cuttings of Gangotri valley (Table 7.1, 7.2). The result obtained in the present case related to number of root per shoot showed that the various concentrations of plant growth hormones were found highly significant (p<0.05) for both male and female cuttings of the target species. It was observed that the number of roots per shoot was found maximum in the cuttings, which were treated with NAA 25 ppm while, the minimum number was observed in the 0 ppm and 0 ppm of IAA (Table 7.3, 7.4). On an average there were 3 to 5 roots formed in a shoot besides, the number of roots per shoot of female cutting was found higher as compared to male. Shuhua et al. (1989) also measured the same number of average roots in their experiment. On the other hand, the type of rooting media applied also affected the mean number of roots as the result revealed which increased the root number of male and female cuttings under composite media (Table 7.4). The cuttings treated with different concentrations of IAA, IBA and NAA showed the significant differences (p<0.05) for the root length of male and female cuttings treated with both the media. The results revealed that the IBA ppm served as a best medium to stimulate and enhance root length under both of the rooting media (Table 7.5, 7.6). The over all results showed 3.5 cm to 5 cm mean root length for 90 days old stem cuttings of 104

the target species. Shuhua et al. (1989) observed an average of 3.6 cm long root for the cuttings collected from the base part of Seabuckthorn plant. The earlier work indicated that after a period of 7 months of planting, the root length increased up to more than 15 cm long under various treatments/concentrations of IBA (Sankhyan et al., 2003). The result of the present study also showed profused rooting with higher root length after 6 months of planting (Plate 7.1). The different concentrations of plant growth hormones applied have no significant differences (p>0.05) for increasing the root diameter. However, the male and female cuttings showed significant differences for root diameter. The female cuttings have a slightly more diameter then the male cuttings but have no significant differences (p>0.05) within the two rooting media (Table 7.7, 7.8). The average root diameter of the cuttings of 90 days was measured 2.2±0.2 mm for male and 2.3±0.2 mm for female cuttings of H. rhamnoides. 105

25 ppm 90 80 70 Rooting (%) 60 40 30 20 10 0 Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand IAA IBA NAA Control Fig. 7.1. Effect of 25 ppm of different plant growth hormones on rooting % of male and female cuttings of H. rhamnoides collected from five valleys under both the rooting media (soil, soil: sand) 106

ppm 90 80 70 Rooting (%) 60 40 30 20 10 0 Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand IAA IBA NAA Control Fig. 7.2. Effect of ppm of different plant growth hormones on rooting % of male and female cuttings of H. rhamnoides collected from five valleys under both the rooting media (soil, soil: sand) 107

ppm 80 70 60 Rooting (%) 40 30 20 10 0 Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand IAA IBA NAA Control Fig. 7.3. Effect of ppm of different plant growth hormones on rooting % of male and female cuttings of H. rhamnoides collected from five valleys under both the rooting media (soil, soil: sand) 108

300 ppm Rooting (%) 70 60 40 30 20 10 0 Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand IAA IBA NAA Control Fig. 7.4. Effect of 300 ppm of different plant growth hormones on rooting % of male and female cuttings of H. rhamnoides collected from five valleys under both the rooting media (soil, soil: sand) 109

0 ppm 60 Rooting (%) 40 30 20 10 0 Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand IAA IBA NAA Control Fig. 7.5.Effect of 0 ppm of different plant growth hormones on rooting % of male and female cuttings of H. rhamnoides collected from five valleys under both the rooting media (soil, soil: sand) 110

0 ppm 60 Rooting (%) 40 30 20 10 0 Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand Soil Soil: Sand IAA IBA NAA Control Fig. 7.6. Effect of 0 ppm of different plant growth hormones on rooting % of male and female cuttings of H. rhamnoides collected from five valleys under both the rooting media (soil, soil: sand) 111