Standardization of vegetative propagation in Terminalia chebula Retz. for germplasm conservation

Abstract

Terminalia chebula Retz. is a multipurpose tree but the primary purpose of cultivating and raising this tree species is its fruits. Large sized fruits easily fetch higher prices in the national and global markets. The availability of superior germplasm is, however, restricted by its very low natural regeneration, poor germination capacity of the seeds, very little knowledge about its propagation techniques, long juvenile period for fruits production which is almost 15–20 years. The availability of superior germplasm and shortening of long juvenile period can be resolved by the adoption of various vegetative propagation techniques. In the present study, scions of T. chebula Retz. were grafted on three different rootstocks; T. chebula Retz., Terminalia bellirica Roxb. and Terminalia arjuna Bedd to standardize vegetative propagation techniques in T. chebula. The grafting and budding methods used were cleft grafting, side-veneer grafting and patch budding. The results revealed that out of all the propagation techniques evaluated under nursery conditions, cleft grafting was most promising. It showed better results on principal parameters like graft survival ratio (46.67) and graft take ratio (60.00). In case of rootstocks, T. arjuna performed well on most of the growth parameters. Hence, it is concluded that T. arjuna as a rootstock can be cleft grafted with T. chebula Retz. scion not only to obtain healthy plants with desired characteristics in a short time but also to conserve its germplasm.

Introduction

Terminalia chebula Retz. commonly known as Harad, is a multipurpose tree belonging to family Combretaceae. In India, it is widely distributed in mixed dry deciduous forests and can be found frequently in tropical and subtropical zones, mostly in hilly tracts. It is a multipurpose species but considered primarily for its fruit which possess multitudinous medicinal properties. Terminalia chebula Retz. is an important component of Triphala, the famous polyherbal formula of Ayurveda, it plays a crucial role for maintenance of various diseases and oral health (Bhuvaneswari et al., 2020).The demand of Harad due to the medicinal properties is recognizable as major industries like Dabur, Kapiva, Badiyanath, Himalaya, Organic India use it for manufacturing their products (Singh et al., 2020).

There is huge demand of Harad in national as well as international market. Being a global dominant producer, India exports almost 20% of its total production (Saleem et al., 2013). However, there are some problems associated with the species i.e. the scale of natural regeneration of T. chebula Retz. is miniscule, its seeds have poor germination capacity, trees have long juvenile period and there is lack of availability of superior germplasm. If these problems are solved one can scale up production and amplify net returns (Singh et al., 2021). Thus, the present experiment was carried out to produce superior Harad clones via vegetative propagation techniques.

Material and methods

The present experiment entitled ‘Standardization of vegetative propagation in Terminalia chebula Retz. for germplasm conservation’ was carried out in experimental farm of Division of Agroforestry, Sher-e-Kashmir University of Agricultural Sciences and Technology, Jammu, Chatha, J&K, India 180,009 for the period of 90 d from March to June, 2022. The objective of the investigation was to standardize vegetative propagation technique and rootstock for successful propagation of T. chebula Retz. Cleft grafting (V₁), side veneer grafting (V₂) and patch budding (V₃) were the methods of propagation used whereas three different rootstocks were T. chebula Retz. (R₁), Terminalia bellirica Roxb. (R₂) and Terminalia arjuna Bedd. (R₃). Scion wood used for grafting operations was collected from the superior mother plants of Harad planted in the experimental farm itself. The design of experiment was Two Factorial CRD with nine treatment combinations and the number of replications were three. The observations recorded were; number of days taken to bud sprouting, graft take ratio (%), graft survival ratio (%), graft shoot diameter (mm), number of shoots per plant, number of leaves per plant, shoot length above graft union (cm), total shoot length (cm), shoot biomass (g), length of primary root (cm), number of secondary roots, root biomass (g) and root: shoot ratio. The data of different parameters were recorded at different stages of experiment i.e. the data pertaining to number of days taken to bud sprouting and graft take ratio was recorded in initial stages right after the bud break. While for number of shoots per plant, number of leaves per plant, shoot length above graft union (cm) and total shoot length (cm) the data was recorded after 60 and 90 DAG (Days After Grafting) whereas graft survival ratio, shoot biomass (g), length of primary root (cm), number of secondary roots, root biomass (g) and root:shoot ratio was evaluated at the end of vegetative period of experiment (90 DAG).To promote the upright growth lateral shoots arising from the rootstocks were pruned regularly to channelize maximum sap flow toward the scion tissue, constant weeding operations were done. There was a severe impact of heat wave in north India during the time of experiment, to alleviate its impact regular irrigation was done. The acquired data from the present experiment was analysed in statistical package RStudio.

Results

Performance of different rootstocks with respect to the observed parameters

The rootstock had a strong influence on performance of the scion with regard to different parameters (Table 1). R₃ (T. arjuna Bedd.) took the least (6.88) number of days for bud breaking which was statistically superior to R₁ (T. chebula Retz.−8.00 d) and R₂ (T. bellirica Roxb.−8.11 d). Rootstock R₃ (T. arjuna Bedd.) had significantly higher graft take ratio (72.22%) which was statistically at par with the rootstock R₁ (T. chebula Retz. – 70.00%) and superior than root stock R₂ (T. bellirica Roxb. – 11.11%). The scions of T. chebula Retz. grafted on R₂ (T. bellirica Roxb.) failed to survive. The highest survival was observed in R₃V₁ (T. arjuna Bedd.−70% followed by T. chebula- 60%).

Table 1. Performance of different rootstocks with respect to the observed parameters

NODTBS, Number of days taken to bud sprouting; GTR, Graft take ratio; GSR, Graft survival ratio; GSD, Graft shoot diameter; NSPP, Number of shoots per plant; NOLPP, Number of leaves per plant; SLAG, Shoot length above graft union; TSL, Total shoot length; SB, Shoot biomass; LPR, Length of primary root; NOSR, Number of secondary roots; RB, Root biomass; RSR, Root: shoot ratio; R₁, Terminalia chebula Retz.; R₂, Terminalia bellirica Roxb.; R₃, Terminalia arjuna Bedd.

*Figures in parentheses are transformed value.

Graft shoot diameter, number of shoots per plant, number of leaves per plant, shoot length above graft union, total shoot length, shoot biomass, length of primary root, number of secondary roots and root biomass were recorded twice i.e. at 60 d and at 90 d (Table 1). At both 60 and 90 DAG, significant impact of rootstock was observed on graft shoot diameter. In R₃ (T. arjuna Bedd.) maximum graft shoot diameter (60 DAG: 3.70 mm & 90 DAG: 3.88 mm) was recorded which was statistically at par with R₁ (T. chebula Retz.) value (60 DAG: 3.63 mm & 90 DAG: 3.76 mm).

The effect of rootstock on number of shoots was significant. R₃ (T. arjuna Bedd.) had the maximum (60 DAG: 1.26 & 90 DAG: 1.26) number of shoots per plant which was statistically at par with R₁ (T. chebula Retz.) (60 DAG: 1.22 & 90 DAG: 1.22). R₂ (T. belliricaRoxb.) had no shoots with all the three types of grafts.

There was significant effect of rootstock on number of leaves per plant. R₃ (T. arjuna Bedd.) had the maximum (60 DAG: 15.67 & 90 DAG: 21.30) leaves per plant, which was considerably higher than R₁ (T. chebula Retz.) (60 DAG: 12.22 and 90 DAG: 16.04).

The impact of rootstock on the shoot length above graft union was significant. R₃ (T. arjuna Bedd.) had the maximum (60 DAG: 33.55 cm & 90 DAG: 37.26 cm) shoot length above graft union and it was significantly higher than R₁ (T. chebula Retz.) (60 DAG: 32.14 cm & 90 DAG: 34.97 cm).

In case of total shoot length, R₃ (T. arjuna Bedd.) had the maximum (60 DAG: 48.00 cm & 90 DAG: 51.70 cm) total shoot length and it was statistically at par with R₁ (T. chebula Retz.) at 60 DAG (47.03 cm) but superior when compared at 90 DAG (49.86 cm) (Table 1).

The species of rootstock significantly affected the shoot biomass. Maximum shoot biomass was recorded in R₃ (T. arjuna Bedd. 34.27 g) which was statistically superior to R₁ (T. chebula Retz. 28.59 g).

The kind of rootstock affected the number of secondary roots significantly. R₃ (T. arjuna Bedd.) had the maximum (39.45) number of secondary roots which was statistically superior to R₁ (T. chebula Retz. 29.13). The effect of rootstock on root: shoot ratio was significant. R₁ (T. chebula Retz.) had the maximum (1.49) root: shoot ratio which was statistically superior to with R₃ (T. arjuna Bedd.) (0.63).

Performance of vegetative propagation techniques with respect to the observed parameters

There was a significant effect of vegetative propagation methods (Table 2). V₃ (patch budding) resulted into earliest (4.44 d) bud sprouting and was statistically superior to the other propagation techniques. V₁ (cleft grafting) had the highest graft take ratio (60.00%) and it was statistically superior to the other vegetative propagation methods (Fig. 1). The plants with cleft grafting (V₁) showed the highest survival (46.67%) which was statistically at par with V₃ (patch budding – 42.22%) and superior to V₂ (side-veneer grafting – 41.11%).

Table 2. Performance of vegetative propagation techniques with respect to the observed parameters

NODTBS, Number of days taken to bud sprouting; GTR, Graft take ratio; GSR, Graft survival ratio; GSD, Graft shoot diameter; NSPP, Number of shoots per plant; NOLPP, Number of leaves per plant; SLAG, Shoot length above graft union; TSL, Total shoot length; SB, Shoot biomass; LPR, Length of primary root; NOSR, Number of secondary roots; RB, Root biomass; RSR, Root: shoot ratio; V₁, Cleft grafting; V₂, Side−veneer grafting; V₃, Patch budding.

*Figures in parentheses are transformed value.

Figure 1. Cleft grafting of harad using Terminalia arjuna as rootstock.

The effect of method of vegetative propagation on graft shoot diameter, number of shoots per plant, number of leaves per plant, shoot length above graft union, total shoot length, shoot biomass, length of primary root, number of secondary roots and root biomass were recorded twice i.e. at 60 d and at 90 d.

V₃ (Patch budding) showed highest value (60 DAG: 2.78 mm & 90 DAG: 2.88 mm) and turned out to be statistically superior to other propagation techniques (Fig. 2). V₁ (cleft budding) showed highest value (60 DAG: 1.03 & 90 DAG: 1.03) and turned out to be statistically superior to other propagation techniques. The highest number was observed in cleft grafting (V₁) (60 DAG: 11.33 & 90 DAG: 15.23) followed by V₃ (60 DAG: 8.64 & 90 DAG: 11.37) and V₂ (60 DAG: 7.92 & 90 DAG: 11.07) after 60 d and 90 d respectively. In patch budded (V₃) plants, maximum shoot length above graft union (60 DAG: 27.77 cm & 90 DAG: 29.97 cm) was registered and minimum length (60 DAG: 17.37 cm & 90 DAG: 19.44 cm) was recorded in side-veneer grafting (V₂). The maximum total shoot length was found to be in V₃ (patch budding) (60 DAG: 39.22 cm & 90 DAG: 41.41 cm) followed by V₁ (60 DAG: 29.66 cm & 90 DAG: 31.92 cm) and V₂ (60 DAG: 26.14 cm & 90 DAG: 31.92 cm). Propagation through cleft grafting (V₁) outperformed among the three methods with the maximum value (23.70 g) which is statistically superior to V₂ (side-veneer grafting 19.26 g) and V₃ (patch budding 19.88). The length of primary root was found to be maximum in cleft grafted plants (V₁) (17.66 cm). It was statistically at par with V₂ (16.64 cm) and V₃ (16.59 cm). V₁ (cleft grafting) performed significantly better in case of number of secondary roots with the maximum value (24.19) which was statistically superior to V₂ (side-veneer grafting) (Fig. 3) and V₃ (patch budding). V₃ (patch budding) had the maximum (17.30 g) value which was statistically at par with the remaining vegetative propagation techniques. V₁ (cleft budding) showed highest ratio (0.81) and was statistically superior from other propagation techniques.

Figure 2. Patch budding of harad using Terminalia arjuna as rootstock.

Figure 3. Side veneer grafting of harad using Terminalia arjuna as rootstock.

Performance of treatment combinations with respect to the observed parameters

The interactions between root stock and method of vegetative propagation helped to finally reach to conclusion (Table 3). As regards individual parameter and the interaction, the results are as follows:

Table 3. Performance of treatment combinations with respect to the observed parameters

NODTBS, Number of days taken to bud sprouting; GTR, Graft take ratio; GSR, Graft survival ratio; GSD, Graft shoot diameter; NSPP, Number of shoots per plant; NOLPP, Number of leaves per plant; SLAG, Shoot length above graft union; TSL, Total shoot length; SB, Shoot biomass; LPR, Length of primary root; NOSR, Number of secondary roots; RB, Root biomass; RSR, Root:shoot ratio; R₁, Terminalia chebula Retz.; R₂, Terminalia bellirica Roxb.; R₃, Terminalia arjuna Bedd.; V₁, Cleft grafting; V₂, Side−veneer grafting; V₃, Patch budding; T, Treatment combination.

*Figures in parentheses are transformed value.

The interaction R₃V₃ (T. arjuna Bedd. × patch budding) took the minimum days – 6.33 d for bud sprouting and was statistically at par with R₃V₁ (T. arjuna Bedd. × cleft grafting – 6.66 d) and R₁V₃ (T. chebula Retz. × patch budding – 7.00 d). Interaction, R₂V₂ (T. bellirica Roxb. × side-veneer grafting) took the maximum −13.00 d for bud sprouting, whereas, R₂V₃ (T. belliricaRoxb. × patch budding) failed to sprout. The scions of T. chebula Retz. grafted on R₂ (T. bellirica Roxb.) failed to survive. The highest survival was observed in R₃V₁ (T. arjuna Bedd. × cleft grafting – 76.66%) and minimum in R₁V₃ (T. chebula Retz. × patch budding – 56.66%).

The interaction effect was significant in case of graft shoot diameter. R₃V₃ (T. arjuna Bedd. × patch budding) showed the maximum graft shoot diameter (60 DAG: 4.47 mm & 90 DAG: 4.65 mm) which was statistically superior to the remaining treatment combinations. However, the least graft shoot diameter was recorded in R₃V₂ (T. arjuna Bedd. × side-veneer grafting) (60 DAG: 3.18 mm & 90 DAG: 3.36 mm).

The effect of vegetative propagation method was significantly observed in number of leaves per plant. R₃V₁ (T. arjuna Bedd. × cleft grafting) had the maximum (60 DAG: 19.88 & 90 DAG: 27.25) leaves per plant which was best among all treatments. Whereas, R₁V₂ (T. chebula Retz. × side-veneer grafting) had the minimum (60 DAG: 10.77 & 90 DAG: 14.11) number of leaves per plant.

The shoot length above graft union was significantly affected both by rootstock and the propagation method performed over it. R₃V₃ (T. arjuna Bedd. × patch budding) exhibited the maximum (60 DAG: 42.66 cm & 90 DAG: 46.00 cm) shoot length above the graft union and performed statistically superior among all other treatment combinations. However, the least (60 DAG: 24.78 cm & 90 DAG: 28.11 cm) shoot length above graft union was recorded in R₃V₂ (T. arjuna Bedd. × side-veneer grafting).

The interaction affected the total shoot length significantly. R₃V₃ (T. arjuna Bedd. × patch budding) recorded the maximum (60 DAG: 59.00 cm & 90 DAG: 62.33 cm) shoot length and it was statistically at par with R₁V₃ (T. chebula Retz. × patch budding) (60 DAG: 58.67 cm & 90 DAG: 61.92 cm). The least total shoot length was recorded in R₃V₂ (T. arjuna Bedd. × side-veneer grafting) (60 DAG: 38.11 cm & 90 DAG: 41.44 cm).

The interaction also significantly affected the shoot biomass. Maximum shoot biomass (40.45 g) was noticed in treatment combination R₃V₁ (T. arjuna Bedd. × cleft grafting) and it was statistically superior than the remaining interactions. The minimum (26.92 g) shoot biomass was observed in R₁V₂ (T. chebula Retz. × side-veneer grafting).

Though the interaction had non-significant effect on number of primary roots and root biomass and root shoot ratio, but in case of secondary roots, maximum number of secondary roots (42.44) was observed in treatment combination R₃V₁ (T. arjuna Bedd. × cleft grafting) and it was statistically significant than the remaining combinations. However, the minimum number of secondary roots were recorded in R₁V₂ (T. chebula Retz. × side-veneer grafting 27.81) (Table 3).

Discussion

The basic purpose of conducting this study was to identify the best rootstock and vegetative technique for propagation of Harad. Although, the vegetative propagation techniques using harad as rootstock had already been reported by Saleem et al. (2013) but the germination percentage of harad fruits is very low (20–30%) compared to Terminalia bellerica (upto 50%) and T. arjuna (upto 90%). Besides, Harad is slow growing compared to other two species. Identification of suitable rootstock and vegetative techniques would help in mass multiplication of superior strains and their conservation.

The effect of rootstock was significant in most of the observations except graft survival ratio, length of primary roots and root biomass. R₃(T. arjuna Bedd.) performed well when compared to R₁(T. chebula Retz.) with higher values in major and essential parameters like; number of days taken to bud sprouting (6.88), graft take ratio (72.22%) etc. (Table 1). Whereas R₂ (T. bellirica Roxb.) failed miserably to survive the experiment. It might be due to high temperature and low humidity (35.4°C and 22%) in the third week of March, T. bellerica requires mean annual temperature in the range of 22–28°C and annual rainfall in the range of 900–1300 mm for its best growth. Although in few plants bud breaking was reported but it might be due to the reserve food material present in the scion tissue. Later on the sprouted buds turned yellow and perished, thus no further readings were recorded for Terminalia bellerica as rootstock. The performance of R₃ (T. arjuna Bedd.) was better among all and it might be due to the better root morphology when compared to R₁ (T. chebula Retz.) The number of secondary roots in R₃ (39.45) were significantly higher compared to R₁ (29.13). On ocular basis it was observed that the R₃ was having denser fibrous root structure when compared to R₁. Also, there was presence of fine root hairs over tertiary roots of grafted R₃ which was absent in R₁ and control R₃. Root branching, its development during the crop period and penetration in soil might have resulted in better plant growth and stock scion union. Similar findings have been reported by Wang et al. (2006) regarding the role of root morphology in nutrient uptake. Zhu et al. (2021) has also stated that fine root hairs play better role in belowground ecosystem which in turns helps greatly in nutrient cycling with in the plant. Thus it can be concluded that better root morphology of T. arjuna Bedd. and development of fine root hairs might have amplified the nutrient cycling and resulted in overall better growth of scion tissue when grafted on R₃ which in turn resulted in development of healthy plants.

The effect of vegetative propagation techniques was significant in all parameters. V₁ (cleft grafting) showed promising results in most of the desired parameters like; graft take ratio (60.00), graft survival ratio (46.67) etc. (Table 2) but the earliest bud break was recorded in the V₃ (Patch Budding). Both V₁ and V₃ performed well in said parameters and can be recommended but the survival and take ratio was higher in the V₁. Rahayu et al. (2020) had stated that success of graft depends on cambium fusion. In cleft grafting the exposed area of cambium is much larger compared to other methods, also the interlocking tongues of rootstock provided natural pressure which play an important role in union formation. Similar results regarding cleft grafting have been observed by Singh et al. (2019) in Juglans regia L. and Tripathi and Karunakaran (2019) in Persea americana Mill.

Out of all treatment combinations, T₇ (Cleft grafting on T. arjuna Bedd.) showed promising results (Table 3). From the present nursery studies, it has been found that T. arjuna Bedd. rootstock cleft grafted with superior Harad scion wood resulted into healthy plants with desirable characteristics. Further on farm trials and extensive experimentation is required to prove the success of this intra species grafting involving T. chebula and T. arjuna.