The Leafy-Stem-Buried Etiolation Contributed to the High Efficiency of Rootstock Vegetative Propagation in Avocado ( Persea americana )

: Because of their tolerance to root rot and many abiotic stresses, rootstocks are generally required for commercial avocado production. However, cutting and air-layering, which are popular methods of vegetative propagation for producing large numbers of uniform and genetically identical plants, have been unsuccessful for years. To develop a practical and efficient rooting procedure for selected avocado clonal rootstocks, the present research investigated the effects of various factors on rooting cutting. Shoots were divided into three groups (air layering, direct cutting, and stem-buried etiolation) and treated with different combinations (plant growth regulators, PGRs; soaking time; and culture media), in which orthogonal or randomized-block designs were used. The rooting rate, average root length, average root number, average root dry weight, and rooting quality (Q value) were used as evaluation indicators. The results show that etiolation treatment of the mother shoot is the requisite condition for avocado shoot rooting. In addition, the rooting effect of etiolated cuttings was strongly impacted by PGR type, concentration, and soaking time, whereas no significant differences were detected among the culture media. Among these factors, the roots and the survival rates of etiolated cuttings of two hard-to-root varieties were optimal under the following conditions: indolebutyric acid concentration of 2500 mg/L, dipping duration for 5 s, and perlite:vermiculite culture at 1:1. The rooting rate of ‘Dusa’ was generally greater than that of ‘Duke 7’ and reached 82%.


Introduction
Despite receiving widespread attention from an increasing number of people for just over 100 years, avocado has attracted increased interest around the world due to its unique nutritional value.In the past few years, the production and consumption of avocado have dramatically increased [1].It is one of the highest value crops per acre [2].The creamy consistency of avocado makes it one of the first fresh fruits that a baby can eat [3].The United States, Japan, Europe, and South America all value avocado as a fruit, with features of fruit, grain, and oil [4,5].The most important losses worldwide are due to Phytophthora root rot caused by Phytophthora cinnamomi [6].Avocado is also considered one of the most sensitive crops to abiotic stresses, such as salt, cold, and alkaline conditions [2].Elite resistant rootstock, a common concept used worldwide [7,8], is the major basis for counteracting these adverse effects [9].
Avocado rootstock today is most often propagated from seeds.This production system has multiple problems, such as insufficient seeds produced by many good germplasm lines, and great genetic variation.Approximately half a century ago, successful avocado rooting of shoots using etiolation was reported [10,11], as were grafting and air-layering techniques [12,13], while propagation by cuttings and air-layering has not been successful in a few areas [14].These different results might be attributed to difference in genotype, maternal tree age, cutting collection time, cutting type, environment, and technical details.In addition to the feasibility of avocado cuttings and air-layering in previous reports, the techniques themselves were too practical complex.As mentioned by Duman et al. [5], the desired rootstock is grafted onto a nurse seedling rootstock, which is grown in the absence of light to develop an etiolated branch; then, the new branch is partially covered with soil and induced to root by auxin and is subsequently grafted again as the rootstock of a selected cultivar scion; the new seedling is then disconnected from the nurse rootstock and grown independently.Other methods include only the etiolation step, in which etiolated branches are induced to be rooting but are still connected to the mother plant [15]; thus.this process is also complex.Tissue culture can produce rooted plantlets for some varieties, but it requires equipment and facilities and takes 8-9 months to obtain rooted plantlets, and sometimes the chances of plantlet survival are small [16].In fact, the lack of availability of many good resistant rootstocks has greatly restricted the extension of new excellent avocado varieties worldwide.
In this study, two good rootstock varieties, 'Duke 7' (which has good resistance to root rot) [17] and 'Dusa' (which has good resistance to cold, salt, and alkali conditions), were used as materials.The aim of this study was to develop a readily applicable technique in avocado that permits the reproduction of large numbers of single selections, and, thus, provides a theoretical and technical reference for the exploitation and application of highly resistant avocado rootstocks.

Plant Material and Orchard Information
Two varieties, 'Duke 7' (with good resistance to root rot) and 'Dusa' (with good resistance to salt), were selected.They were grown in the avocado germplasm repository, which is located at the South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences, Zhanjiang, China.The soil of the experimental field was red and yellow loam.Both varieties were grafted onto 10-year-old 'Hass' rootstock and subjected to the same cultivation and management conditions.Trees of similar size and vigor were used.

Overall Experimental Design and Treatments
After a series of preliminary trials, three experiments were conducted from March to July 2018 in the present study.They were called as air-layering, direct cutting, and stem-buried etiolation.The first experiment was performed in trees for three months and then continued in a greenhouse (the layering shoots were cut and moved to the greenhouse for further culture); the latter two were conducted in the greenhouse, with an average of 25 • C and >70% humidity (recorded by temperature and humidity recorders, ZDR-20, Hangzhou Zida Instrument Co., Ltd., Hangzhou, China Figure 1).The entire experimental design is graphically described in Figure 2. All treatments were repeated three times with a randomized block and/or orthogonal experimental design.

Air-Layering Experiment
For each variety, nine trees were used which were further divided into three groups (three replications).For each tree, more than ten woody shoots (diameter 1.5-2.5 cm) were chosen.For each shoot, a ring of bark was removed near the base.Two days later, the wound was covered with medium and then bagged with plastic and rope (Figure 3A).The medium consisted of the local red clay loam that turned into mud by mixing with 100 mg•L −1 NAA or 500 mg•L −1 IAA or 2500 mg•L −1 IBA solution.When many callus had grown from the bark end (Figure 3B) about three months later, we truncated the shoots at their base from the trees, pruned their leave and small branches, then moved them to the greenhouse and planted in sand-pots for further cultivation (Figure 3C).Foliar fertilizer was applied once per week, and insecticide was sprayed every 3 weeks.

Air-Layering Experiment
For each variety, nine trees were used which were further divided into three groups (three replications).For each tree, more than ten woody shoots (diameter 1.5-2.5 cm) were chosen.For each shoot, a ring of bark was removed near the base.Two days later, the wound was covered with medium and then bagged with plastic and rope (Figure 3A).The medium consisted of the local red clay loam that turned into mud by mixing with 100 mg•L −1 NAA or 500 mg•L −1 IAA or 2500 mg•L −1 IBA solution.When many callus had grown from the bark end (Figure 3B) about three months later, we truncated the shoots at their base from the trees, pruned their leave and small branches, then moved them to the greenhouse and planted in sand-pots for further cultivation (Figure 3C).Foliar fertilizer was applied once per week, and insecticide was sprayed every 3 weeks.

Air-Layering Experiment
For each variety, nine trees were used which were further divided into three groups (three replications).For each tree, more than ten woody shoots (diameter 1.5-2.5 cm) were chosen.For each shoot, a ring of bark was removed near the base.Two days later, the wound was covered with medium and then bagged with plastic and rope (Figure 3A).The medium consisted of the local red clay loam that turned into mud by mixing with 100 mg•L −1 NAA or 500 mg•L −1 IAA or 2500 mg•L −1 IBA solution.When many callus had grown from the bark end (Figure 3B) about three months later, we truncated the shoots at their base from the trees, pruned their leave and small branches, then moved them to the greenhouse and planted in sand-pots for further cultivation (Figure 3C).Foliar fertilizer was

Direct Cutting Experiment
For each variety, more than nine well-grown mature trees were selected for sampling shoot cuttings that had a diameter of 0.8-1.5 cm and length of 20-25 cm.More than 370 cuttings (leaves removed) were obtained.A four-factor three-level L9(3 4 ) orthogonal design was used to reveal the effects of different factors and their levels on cutting rooting (Table 1).In the step of soaking with auxin solution, cuttings were laid out vertically and only the bases (1 cm height) were soaked.The cuttings were directly inserted into the media (Figure 4(A1)).Foliar fertilizer was applied once per week, and insecticide was sprayed every 3 weeks.

Direct Cutting Experiment
For each variety, more than nine well-grown mature trees were selected for sampling shoot cuttings that had a diameter of 0.8-1.5 cm and length of 20-25 cm.More than 370 cuttings (leaves removed) were obtained.A four-factor three-level L 9 (3 4 ) orthogonal design was used to reveal the effects of different factors and their levels on cutting rooting (Table 1).In the step of soaking with auxin solution, cuttings were laid out vertically and only the bases (1 cm height) were soaked.The cuttings were directly inserted into the media (Figure 4(A 1 )).Foliar fertilizer was applied once per week, and insecticide was sprayed every 3 weeks.

Levels Factors PGR (A) Concentration of PGR (B) Culture Medium (C) Soaking Time (D)
Horticulturae 2024, 10, x FOR PEER REVIEW 5 of 13

Stem-Buried Etiolation Experiment
Lignified shoots (leafy stems) with an approximate diameter of 2 cm and length of 20-25 cm were cut from each variety.The four-factor three-level L9(3 4 ) orthogonal design used was the same as that used for the direct cutting experiment.The factors were A (PGR type), B (PGR concentration), C (culture medium), and D (soaking time), and their corresponding levels are shown in Table 1.The entire cutting was horizontally buried in the medium to a depth of 15-20 cm.At the first sign of budding (growth from mother cutting and appearance above ground), foliar fertilizer was applied once per week, and insecticide was applied every 3 weeks (Figure 4(B1,B2)).
When the new buds had more than 6 leaves, the plantlets in each treatment were divided into two groups.Half of the cuttings were maintained in media for continuing growth of the buds (Figure 4(B2-B4')), which was referred to as in situ rooting.The other half of the cuttings were dug and the etiolated buds were detached at the base (to separate from the mother cutting), most of the leaves were pruned, and the cuttings were finally

Stem-Buried Etiolation Experiment
Lignified shoots (leafy stems) with an approximate diameter of 2 cm and length of 20-25 cm were cut from each variety.The four-factor three-level L 9 (3 4 ) orthogonal design used was the same as that used for the direct cutting experiment.The factors were A (PGR type), B (PGR concentration), C (culture medium), and D (soaking time), and their corresponding levels are shown in Table 1.The entire cutting was horizontally buried in the medium to a depth of 15-20 cm.At the first sign of budding (growth from mother cutting and appearance above ground), foliar fertilizer was applied once per week, and insecticide was applied every 3 weeks (Figure 4(B 1 ,B 2 )).
When the new buds had more than 6 leaves, the plantlets in each treatment were divided into two groups.Half of the cuttings were maintained in media for continuing growth of the buds (Figure 4(B 2 -B 4 ')), which was referred to as in situ rooting.The other half of the cuttings were dug and the etiolated buds were detached at the base (to separate from the mother cutting), most of the leaves were pruned, and the cuttings were finally transplanted to sandy pots with sandy soil (Figure 4(B 2 -B 5 )).This group was called as re-cutting rooting, or this group was referred to as re-cutting.

Evaluation of the Rooting Quality Index
The rooting rate was calculated as the percentage of (rooting cuttings)/(total cuttings).The root length was measured with a Vernier caliper.The root systems were dried in a blast drying oven (DHG-9240A, Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China) and then weighed (ME104, Mettler Toledo, Shanghai, China).The rooting quality (Q value) was comprehensively evaluated as described by Zhou et al. [18]: Q = (rooting rate × 50%) + (average root number × 25%) + (average root length × 25%).

Statistical Analysis
Duncan's multiple range test, range analysis, variance analysis, calculation of means and standard deviations, and plotting were performed using SPSS 17.0 and Excel 2010 software.

Air Layering and Direct Cutting
Although one shoot survived after air layering and cultivation in a pot, the root system was thin and frail and lost viability during the transplanting process (Figure 3C-H).In the direct cutting experiment, hundreds of cuttings from different treatment groups were inserted into media.One to three months later, the whole cuttings were gradually withered, and no roots were successfully induced in any of the treatments (Figure 4(A 1 ,A 2 )).These results suggest that both air layering and direct cutting are unsuitable for vegetative propagation of the two varieties tested, which, in turn, implies that avocado rooting requires stimulation from particular substances or environmental conditions, or that the presence of certain inhibitors suppresses rooting.

Overview Results of Stem-Buried Etiolation
After the cuttings were completely embedded in media and treated in the shade for two months, most of the buried mother shoots gave rise to more than one etiolated bud that broke the ground and became new shoots.The new shoots of 20 cm in length showed pronounced etiolation up to 5 cm above the base (the joint location of the bud and mother cutting).From then on, for each treatment, the budded cuttings were divided into two groups, i.e., half of which were left in place and cultured to allow rooting (group of in situ rooting) (Figure 4(B 2 -B 4 ')), the rest of the cuttings were severed at the new shoot base, and the shoots were resubjected to root induction in soil (group of re-cutting) (Figure 4(B 2 -B 5 )).Unexpectedly, both groups exhibited outstanding results for rooting and survival (Figure 4(B 4 ',B 5 )).The results indicate that stem-buried etiolation is effective for the propagation of avocado rootstocks.In addition, 'Dusa' had higher rooting rates than 'Duke 7' for both groups (Supplementary Tables S1-S4), suggesting that rooting capability varied greatly between the varieties.

Rooting Rate of Stem-Buried Etiolation
The rooting rate is one of the most important indicators for evaluating crop rooting quality.When cuttings were treated with the number 9 combination (A 3 B 3 C 2 D 1 ), 'Dusa' in situ rooting treatment reached 82% (Supplementary Table S1), which was the highest value among all treatments in all groups.There was no significant difference between any two treatments in the 'Duke 7' in situ rooting group (Supplementary Table S2).For the A 2 B 1 C 2 D 3 combination, rooting could only be induced in the 'Duke 7' in situ rooting group (Supplementary Table S2, Figure 5A).To test whether rooting could be induced only by etiolation, cuttings without PGR treatment were buried only by C 1 , C 2, or C 3 in the summer seasons of the following two years (2019 and 2020).Unexpectedly, 0-5 cuttings could generate buds and roots in each treatment in any year (rooting rate < 16.7%), suggesting that etiolation itself could slightly induce bud and root growth.
Horticulturae 2024, 10, x FOR PEER REVIEW 7 of 13 seasons of the following two years (2019 and 2020).Unexpectedly, 0-5 cuttings could generate buds and roots in each treatment in any year (rooting rate < 16.7%), suggesting that etiolation itself could slightly induce bud and root growth.   1 (the same below).

Average Length and Average Dry Weight of Roots of Stem-Buried Etiolation
No significant difference in average root length was found among the four samples within any one of the nine combinations (Figure 5B).
After treatment with the A2B1C2D3 combination, the average root dry weight of the 'Duke 7' in situ rooting group was 0.36 g, while no roots were generated in the other three groups.Except for this combination, the other combinations showed no significant differences among the four groups (Figure 5C).
and A 3 B 3 C 2 D 1 treatments, respectively, as listed in Table 1 (the same below).

Average Length and Average Dry Weight of Roots of Stem-Buried Etiolation
No significant difference in average root length was found among the four samples within any one of the nine combinations (Figure 5B).
After treatment with the A 2 B 1 C 2 D 3 combination, the average root dry weight of the 'Duke 7' in situ rooting group was 0.36 g, while no roots were generated in the other three groups.Except for this combination, the other combinations showed no significant differences among the four groups (Figure 5C).

Average Root Quantity of Stem-Buried Etiolation
For the A 3 B 3 C 2 D 1 combination, the average root quantities of the 'Dusa' in situ rooting, 'Duke 7' in situ rooting and 'Dusa' re-cutting groups were notably greater than those of the 'Duke 7' re-cutting group.After treatment with the A 2 B 1 C 2 D 3 combination, rooting occurred only in the 'Duke 7' in situ rooting group, with an average root number of 3.5.The other combinations showed no significant differences among the four groups (Figure 5D).

Rooting Quality of Stem-Buried Etiolation
When cuttings were treated with the A 3 B 3 C 2 D 1 combination, the 'Dusa' in situ rooting group showed the greatest rooting quality, which was markedly greater than that of both the re-cutting groups and the 'Duke 7' in situ rooting group.For the A 1 B 2 C 2 D 2 combination, the value of the 'Dusa' in situ rooting group and both re-cutting groups were notably greater than that of the 'Duke 7' in situ rooting group.No significant difference among the four groups of samples was observed for the other combinations (Figure 5E).

Multiple Difference Analysis for Different Factors and Levels in 'Stem-Buried Etiolation' Treatment
The rooting rates of 'Dusa' and 'Duke 7' after in situ cultivation were 0.0-82.0%(Supplementary Table S1) and 6.6-56.7%(Supplementary Table S2), respectively; the corresponding rooting rates after re-cutting were 0.0-63.3%and 0.0-36.7%(Supplementary Tables S3 and S4), respectively.Range analysis reveals that for both in situ rooting and re-cutting, the most important factor affecting the rooting rate of both varieties is the PGR concentration (Supplementary Tables S1-S4).Multiple significance analysis (Figure 5A) and range analysis (Supplementary Tables S1-S4) indicates that the rooting rates of in situ cultivation and re-cutting for both varieties are greatest for the factor level combination A 3 B 3 C 2 D 1 , which is markedly superior to the other combinations.
The average root length of each stem-buried etiolation treatment was within 2.8-11.3cm (Supplementary Tables S1-S4).The average root number reached six in some groups, but for the majority of the shoots, this value ranged from three to five (Supplementary Tables S1-S4).The analysis results indicate that only a certain combination but not any factor or level has a great impact on these two indicators.
The average root dry weight for the stem-buried etiolation treatments ranged from 0.21-0.60g (Supplementary Tables S1-S4).Similar to the responses of average root length, after stem-buried etiolation, the average root dry weight for the 'Dusa' and Duke re-cutting groups and the 'Dusa' in situ rooting group was mostly affected by PGR soaking time (Supplementary Tables S1, S3 and S4).Only the 'Duke 7' in situ rooting group was influenced by PGR type (Supplementary Table S2).All four groups of both varieties achieved the greatest average root dry weight with the A 3 B 3 C 2 D 1 combination, and the differences among the other combinations were not significant within each group (Supplementary Tables S1-S4).
The rooting quality of the 'Duke 7' in situ rooting group was affected mostly by the PGR type (Supplementary Table S2), while the other three groups were affected mostly by the PGRs soaking time (Supplementary Tables S1, S3 and S4), which was similar to the average length and average dry weight of the roots.The four treatment groups of both varieties exhibited optimal rooting quality with the A 3 B 3 C 2 D 1 combination.Moreover, excepted for the 'Duke 7' re-cutting group, the A 3 B 3 C 2 D 1 in the remaining three groups performed markedly better than the other combinations (Supplementary Tables S1-S4).

Discussion
Studies of vegetative propagation, especially of cutting propagation technology, are not only essential and effective for expediting propagation and establishing ex situ conservation bases, but also important means for offspring generations to retain good genetic characteristics of sampled trees [19].Therefore, the establishment of a vegetative propagation system for avocado rootstocks with excellent resistance is highly important for the preservation, development, and utilization of avocado germplasm resources.
In addition to contrasting results [20], researchers worldwide have shown that etiolation can significantly improve the rooting rate and quality of cuttings, such as those of yellowhorn [21], Arabidopsis [22,23], tetraploid black locust [24,25], currant and goose-berry [26], and large tooth maple [27].Our results are, therefore, in line with those of previous studies.
There have been few studies on the relevant mechanisms of rooting cuttings, but chlorophyll is generally absent, the number of parenchyma cells is increased [5,28], and the levels of root-promoting factors (IAA and ABA) are elevated in etiolated buds.These conditions are highly important for the study of cutting methods [29].Based on studies of Arabidopsis, Verstraeten et al. [22] proposed that etiolated hypocotyls express a novel auxinregulated signal transduction pathway, in which the auxin response factors, microRNAs, and environmental conditions that stimulate adventitious roots are integrated.Lu et al. [24] performed physiological and biochemical analyses of the rooting process and reported that etiolated buds contained more endogenous hormones that promoted adventitious root formation than the non-etiolated buds.In addition, the activity of oxidases and the levels of IAA and ABA were high, indicating that etiolated cuttings promoted adventitious root formation by regulating the activities and levels of oxidases and endogenous hormones.
Many exogenous PGRs have crucial impacts on in vitro culture and rooting.Our study shows that etiolation can slightly induce cutting rooting (etiolation without PGR experiments performed in 2019 and 2020), while application of the correct IBA concentration greatly increases the rooting rate (Supplementary Tables S1-S4); these finding are similar to those of many previous studies, such as those of Cutting and Vuuren [16] and Manish et al. [30].Multiple range tests on the orthogonal design data of the stem-buried etiolation group demonstrated that (i) the hormone type, concentration, and soaking time were the most important factors affecting the rooting quality of avocado and that (ii) the same sample group showed marked differences with different hormone concentrations and soaking times.Therefore, if etiolation provides avocado cuttings with the most substantial guarantee for rooting, exogenous hormones and soaking time are the key conditions for improving the rooting rate and quality.A recent study also suggested that etiolation treatment provided a basis while other conditions led to fine tuning for avocado adventitious root formation [5].
Auxin-like compounds, such as indoleacetic acid (IAA), indolebutyric acid (IBA), naphthaleneacetic acid (NAA), and their derivatives play critical roles in rooting induction in cuttings [20].However, the formation of adventitious roots on cuttings was usually regulated not only by auxin [31,32].The air layering and direct cutting treatments in our experiment included auxin screening, and they failed to induce rooting of the avocado roots, confirming the previous conclusion that the formation of adventitious roots was not regulated by auxin alone.Many modern experiments and propagation practices have also validated the fact that auxin is not the only substance that promotes rooting of cuttings, and it must be supplemented by a special class of substances produced in the buds and leaves to stimulate the formation of adventitious roots.These substances are the root-promoting factors, and some reports refer to them as growth enhancers [33], including the C/N ratio, total carbohydrates, total indoles, IAA, IAA/GA ratio, and total phenols, and so on [20].
Some researchers believe that the root-promoting factors alone have no influence on rooting, but they enhance the promoting effect of auxin on rooting [33].For different tree varieties, the rooting of cuttings fails even if auxin is applied.Although this aspect has not been fully studied, some researchers have long believed that root-inhibiting substances exist in cuttings of hard-to-root cultivars [33].In addition, García-Gómez et al. [34] carried out micro-cutting studies on avocado, and discovered that IAA was not essential for the initiation or continued development of root primordia.In the present study, this conclusion was confirmed by the fact that the rooting rate and quality of avocado in the IAA treatment groups did not improve.Studies have shown that rooting is smoother in cultivars that have a higher starch content than in those with a high tannin content, while those with low starch and high tannin content exhibit poor rooting.The effect of auxin is not satisfactory in some plant species, such as for persimmon, peach, and plum.However, Izhaki et al. [35] reported that clone genotype significantly affected the rooting rate, which ranged from 0 to nearly 100% in various clones.The rooting rates of cuttings collected from different positions on the mother plant shoots did not differ significantly, suggesting that juvenility does not play an important role in rooting of D. virginiana cuttings, as not all of the tree varieties exhibit facile rooting with juvenile cuttings.Therefore, it is more objective to evaluate the relationship between rooting and hormones using a comprehensive balance of multiple hormones.Are root-promoting factors and rooting inhibitors present in avocado cuttings?If yes, how do they function?How does the etiolation treatment affect this process?These questions call for further in-depth investigations.
Multiple range tests on the orthogonal design data of the stem-buried etiolation group show that the effect of media composition on the rooting indicators of avocado is not as important as hormone type, concentration, or soaking time.Vermiculite is a rare silicate mineral that is natural and non-toxic, and expands at high temperature.It is produced during the hydration of granite, is capable of ion exchange, and is very effective in improving soil.It can also consistently provide water and minerals necessary for plant growth from the early stage of growth, maintain the stability of temperature for roots, and promote the rapid growth of plants.Perlite is more effective at increasing permeability and water absorption of the nutrient media.The rooting rate of each group was highest for the vermiculite:perlite ratio of 1:1, indicating that this composition provided optimal growth conditions for the frail nascent root system of avocado.
The successful construction of a vegetative propagation system for quality avocado rootstocks in this study lays the technical foundation for the advance and promotion of vegetative propagation of avocado rootstocks.However, the reasons for the difficulty in avocado rooting and survival after direct cutting; the specific conditions necessary for rooting and survival provided by etiolation treatment; the effects of tree age, age of shoots, maturity of cuttings, and number of leaves; and the different technical parameters required by different varieties remain to be studied.

Summary and Conclusions
In the present study, both air layering and direct cutting were unsuitable for vegetative propagation of avocado, because roots were hardly induced from shoots or leafy-stems without etiolation.In contrast, most of the nine treatments involving leafy-stem-buried etiolation resulted in a high rooting rate and excellent rooting quality.The rooting rate of 'Dusa' was significantly greater than that of 'Duke 7' for most of the treatment combinations.In particular, after etiolation, cuttings of both 'Dusa' and 'Duke 7' exhibited optimal rooting quality and survival rates when the medium ratio was perlite:vermiculite = 1:1, the IBA concentration was 2500 mg/L and the dipping duration was 5 s (A 3 B 3 C 2 D 1 combination).Under these conditions, the rooting rate of 'Dusa' in situ reached 82%.To the best of our knowledge, this etiolation method combined with other treatments is the most practical technique for mass avocado rootstock production.The successful construction of a vegetative propagation system will lay the technical foundation for the popularization of avocado rootstocks.

Horticulturae 2024 , 13 Figure 1 .
Figure 1.Real-time recording of temperature and humidity in the greenhouse.

Figure 2 .
Figure 2. Graphical instructions for the entire experimental design.

Figure 1 .
Figure 1.Real-time recording of temperature and humidity in the greenhouse.

Figure 1 .
Figure 1.Real-time recording of temperature and humidity in the greenhouse.

Figure 2 .
Figure 2. Graphical instructions for the entire experimental design.

Figure 2 .
Figure 2. Graphical instructions for the entire experimental design.

Figure 3 .
Figure 3.The process of the air-layering experiment.(A) A ring bark was removed and bagged with medium at the shoot base; (B) calli formed at the bark end; (C) layering shoots cultivated in sandpots; (D-H) roots could not formed or were thin and frail and ultimately lost viability.

Figure 3 .
Figure 3.The process of the air-layering experiment.(A) A ring bark was removed and bagged with medium at the shoot base; (B) calli formed at the bark end; (C) layering shoots cultivated in sand-pots; (D-H) roots could not formed or were thin and frail and ultimately lost viability.

Figure 4 .
Figure 4. Rooting of avocado shoots in direct cutting and stem-buried etiolation experiments.(A) Direct cutting experiment; (B) stem-buried etiolation experiment; (A1,A2) cuttings were directly inserted into the media, and finally none survived; (B1) cuttings that will be buried in a certain medium to a depth of 15-20 cm were horizontally laid out; (B2) cutting budding; (B3',B4') in situ cuttings have vigorous roots; (B3-B5) new rooting-shoots were cut from the mother shoots and transplanted to soil-pot where they also grew well.

Figure 4 .
Figure 4. Rooting of avocado shoots in direct cutting and stem-buried etiolation experiments.(A) Direct cutting experiment; (B) stem-buried etiolation experiment; (A 1 ,A 2 ) cuttings were directly inserted into the media, and finally none survived; (B 1 ) cuttings that will be buried in a certain medium to a depth of 15-20 cm were horizontally laid out; (B 2 ) cutting budding; (B 3 ',B 4 ') in situ cuttings have vigorous roots; (B 3 -B 5 ) new rooting-shoots were cut from the mother shoots and transplanted to soil-pot where they also grew well.

Figure 5 .
Figure 5. Duncan's multiple significance analysis among four groups of samples at the same treatment levels.(A) Effects of various treatments on rooting rate of cuttings from different group; (B) effects of various treatments on average root length of cuttings from different group; (C) effects of various treatments on average root dry weight of cuttings from different group; (D) effects of various treatments on root number of cuttings from different group; (E) effects of various treatments on rooting quality of cuttings from different group.The different letters above each column indicate significant differences in the same color columns according to Duncan's multiple test at p < 0.05; the bars indicate standard deviations; the numbers 1-9 in the legends indicate the A1B1C1D1, A1B2C2D2, A1B3C3D3, A2B1C2D3, A2B2C3D1, A2B3C1D2, A3B1C3D2, A3B2C1D3, and A3B3C2D1 treatments, respectively, as listed in Table 1 (the same below).

Figure 5 .
Figure 5. Duncan's multiple significance analysis among four groups of samples at the same treatment levels.(A) Effects of various treatments on rooting rate of cuttings from different group; (B) effects of various treatments on average root length of cuttings from different group; (C) effects of various treatments on average root dry weight of cuttings from different group; (D) effects of various treatments on root number of cuttings from different group; (E) effects of various treatments on rooting quality of cuttings from different group.The different letters above each column indicate significant differences in the same color columns according to Duncan's multiple test at p < 0.05; the bars indicate standard deviations; the numbers 1-9 in the legends indicate the A 1B 1 C 1 D 1 , A 1 B 2 C 2 D 2 , A 1 B 3 C 3 D 3 , A 2 B 1 C 2 D 3 , A 2 B 2 C 3 D 1 , A 2 B 3 C 1 D 2 , A 3 B 1 C 3 D 2 , A 3 B 2 C 1 D 3 ,and A 3 B 3 C 2 D 1 treatments, respectively, as listed in Table 1 (the same below).