Effect of Hormones (IBA & IAA) on the Propagation of Himalayan Yew in Pakistan: A Conservation Approach

Himalayan yew (Taxus wallichiana) is one of the endangered medicinal plants species having great importance due to the presence of anticancer drug Taxol. This metabolite is mainly used for the treatment of ovarian, breast, AIDS-related cancers, and other indications. The study being reported here was conducted for the propagation of Himalayan yew by using two different trials of Indole Butyric acid (IBA) and Indole acetic acid (IAA) hormones treatments (2000–7000 ppm) through stem cuttings (140 each). In the same way, 3840 cuttings were treated with IBA and IAA from November 2016 to November 2017 at Lalku valley, Swat, Khyber Pakhtunkhwa (KP), Pakistan.


Conclusion
The present study enhanced the potential of conservation and propagation of T. wallichiana. Hence our study suggests and recommends the application of IBA (7000 ppm) as a better hormone for the conservation and propagation of Himalayan yew. Background T. wallichianais one of the threatened medicinal plants of Himalayan Region (1) commonly known as Himalayan yew and belongs to family Taxaceae (2).Other names given to the species are Banrya in tree forests or cool broad-leaved forests. Due to shade demanding nature, Taxus is usually found in association with large tree species such as Abiespindrow, Betula utilizes Pinus wallichiana, Acer cesium, Rhododendron arboreuand, Quercus semecarpifolia (10).
Taxus is very important to treat cancer and many other diseases like bronchitis, snake bites, epilepsy, asthma, aphrodisiac, internal injuries, scorpion, diabetes, and for the diseases of lungs (10,11).
In Pakistan, lack of awareness, slow growth rate, agriculture, construction, habitat loss, forest res, transformation, grazing, over-harvesting, decoration purposes, medicinal use, accidental mortality, lack of management policies, illicit cutting etc. are major threats to the species (10,12,13). Approximately 10 genera of Taxus are now declining at the Northern Hemisphere in temperate zones (14). During 2001During -2005, Technology, Information, Forecasting and Assessment Council (TIFAC) has reported 45 threatened medicinal plants with speci c recommendation for 7 plants including Taxus enlisted in Convention for International Trade in Endangered Species (CITES) (Appendix II) in 1995 (15,16,17,18,19,20,21).
Recently the existence of Yew is receiving high conservation attention due to the high exploitation rate which has reduced its population by 87% (22,23,24,25). Poor regeneration process, slow growth rate and lengthy seed dormancy period of the species (26) signi cantly contribute to hurdles in its conservation. Vegetative propagation, therefore, could be one of the practical options to enhance its natural regeneration. The Taxus species has high regeneration potential by adventitious rooting of fresh stem cuttings (27). Unlike other Taxus species, T. wallichiana is di cult to root and requires longer time (28). Rooting of Taxus stem cuttings is well documented (29,30). The present experiment was aimed to enhance the potential of conservation and propagation of T. wallichiana using the stem cuttings of mature trees with the application of various doses of IBA and IAA.

Test for normality
Results of Kolmogorov-Smirnov and Shapiro-Wilktests are not signi cant therefore the data ful ll the normally assumption (  The Q-Q plots was used to test the assumptions of normality for all the expected and observed values for the four parameters (number of roots, length of root, number of leaves, and number of sprouts). All the values were plotted on the Q-Q plots on a graph which shows that the data is normal (Fig. 4-7). Table 3 shows that Correlation is signi cant at the 0.01 level (2-tailed). Correlations were done between the growth parameters (number of roots, root length (cm), number of leaves, and number of sprouts).

Multicollinearity
There is strong to moderate correlation was found among number of roots-root length (0.736), number of roots-number of leaves (0.800), number of roots-number of sprouts (0.519). Signi cant correlation was also found among root length-number of leaves (0.718), root length-number of sprouts (0.421). Similarly, there is signi cant correlation among number of leaves-number of sprouts (0.541). Hence data contain no multicollinearity. Since the data ful l all the assumption to apply MONOVA technique for further analysis.
Since the p value is < 0.05 (Table 4) which means different treatment at different level are signi cant to each other. Now to check the performance of different combination, which one performs better than the other, for that post hoc test was applied.   (Fig. 8 to 12).  Table 5).

Discussion
The current study indicates that hormone treatment (IBA & IAA) is the favourable and best option for the conservation and vegetative propagation of Himalayan yew. As our study depicts and recommends IBA the best rooting hormones in comparison to the Indole acitic acid ( IAA). In fact, the auxins application is important to produce roots in fresh stem cuttings as they have been experienced for stimulating secondary plant growth that results in the reserve food material mobilization to the root initiation site (31). Auxins application increases rooting and quality of roots in various tree species (32). It is a welldocumented that the auxins application is important for the roots formation in the cuttings of plant stem as they have been found in stimulating the cambial activity which results in reserve food mobilization to the root initiation site (31; 33).
Also, a successful rooting in juvenile shoot cuttings of T. wallichiana with different auxins has been documented from the other temperate areas as well (34,35,36,38,39) Our results are also similar with Nautiyal et al. (1991) (40), who treated hormones on stem cutting of teak for roots induction and concluded that IBA is the favourable and best auxins. Other scientists (41,44,37,42,45,46,43) also documented that the IBA application play a vital role in the propagation of stem cuttings of Taxus. Their results clearly mentioned that vegetative propagation (in-situ conservation) can improve the number of juvenile's plants of genus Taxus.
Several primary root formations under the in uence of IBA application have been obtained in the present trials (7000 ppm). It must be accepted that a higher root to shoot is satisfactory for achieving a higher survival rate after transplantation of eld. It has also been reported that more than optimums concentration of auxins is toxic to the root regeneration, while theoptimum concentration of hormones is favorable (47,48). In our study we had applied the maximum concentration of 7000 ppm of both the auxins (IBA and IAA) which shows favourable response to the growth of T. wallichiana cuttings.
The differential response to changing concentrations of hormones for survivals of T. wallichiana were noted in the present study could be like the action of increasing Auxins (2000 ppm -7000 ppm). The IBA application may have an indirect effect by increasing the translocation speed and the movement of sugar to the cuttings base which in result stimulate rooting (31).
IBA application in the current study signi cantly increased the survival percentage (number of roots, length of roots, number of leaves, and number of sprouts). Same results were obtained in another study by applying different hormones, for the initiation of a maximum rooting percentage in the juvenile fresh stem cuttings of T. wallichiana; IBA is the effective and best hormone (50). It is now decided that IBA is the best rooting auxins for Taxus species and it was con rmed by many workers (36,29). The time duration of our study was kept 20 weeks after the plantation of the cutting of T. wallichiana. Similarly, the auxins effect on growth and induction of adventitious roots of T. wallichiana cuttings after 4 and ½ months of application/treatment and plant growing. In general, it has been found that among various auxins IBA was the more active for root germination showed the maximum percentage of survival, rooting percentage, percentage of callusing roots per cutting, and length of root per cutting (37,41,42,43,45,46). The researcher studied the application of 1000 ppm and 500 ppm of IBA on the T. wallichiana cuttings and found, IBA 500 ppm showed slightly less response than IBA 1000 ppm. IBA 1000 ppm showed the best rooting response on shoot cuttings of T. wallichiana in the spring season with a rooting percentage (95%) compared to other treatments in other seasons (51). In our study the best response was given by 7000 ppm in case of both IBA and IAA. By looking into other studies, it clears that by increasing the concentration up to 7000 ppm the growth will be high.
By using IBA, with some other cultivar of yew (49) that showed that treatment of hormone was very important for successful conservation and propagation of T. wallichiana. By the treatment of hormones, stem cuttings of some other species were also published (55), their research showed that the best response for rooting due to IBA followed by IAA and NAA in rooting percentage, the cuttings heights, Auxins are involved in the formation of root, cambial cells activation, and lateral bud inhibition. They have been found as naturally existing compounds that promotes the formation of root and synthetic auxins also stimulating the emergence of root on cuttings. It is well documented and veri ed maximum time that auxins applied (naturally or arti cially) is needed for adventitious root initiation on stem cuttings. Initial cells division of roots is dependent on either endogenous or applied auxins (56).
Our ndings are also in similarity with (57) that showed that root development in stem cuttings of T. wallichiana using IBA followed by IAA and NAA and in control where no auxins treatment was given, less root formation was observed in those cuttings, and the lowest survival was found during the observation of cuttings. In this case further the survival becomes very di cult and ultimately the cuttings died. In this respect, our results are similar as obtained by (58 (Fig. 8-12). The lowest survival percentages were noticed by control cuttings in both the trials.

Study Area
Our study was carried out with the aim to enhance the potential of conservation and propagation of the valuable and endangered medicinal plant T. Cuttings were collected from various mature patches of Himalayan yew. The cuttings were brought to the nursery, raised in Lalku Forest Research Station. The length ofthe nal cuttings were kept 7 to 8 inches and 3 to 4 nodes were retained in each cutting. The needles at the basal portion (about 2 cm) of the stem cuttings were removed and sterilized using 2% benlate (fungicide) before planting. The cuttings were dipped in fungicidal solution for 5 minutesand dried for 20 to 25 minutes in an open environment. The dried cuttings were treated with 50% (Water: Ethanol) concentrated solution of the IBA and IAA (2000-7000 ppm) for 5 minutes and planted in the polythene bags containing soil. The soil was prepared by mixing forest soil, sand and agriculture soil ofthe area in1:1:1 and sieved properly before lling into polythene bags (Fig. 2).

Experimental Design
The experiment was carried out in a randomized block design with a factorial treatment's arrangement. First trial treatment was carried out with 7 rows and each row containing 20 cuttings. Total of 140 cuttings were taken in the rst trial of the experiment (n = 140; x 20 cuttings x 1 type of cuttings x 7 IBA treatments, 3 replications) (Fig. 3). Same was repeated for the second trial of IAA treatments 7 rows with 20 cuttings each, so a total of 140 cuttings were used (n = 140; x 20 cuttings x 1 type of cuttings x 7 IAA treatments, 3 replications) as shown in (Table 1)