The Effect of Storage Treatments on the Growth of In vitro Agave

(Agave sisalana L. Perrine) Seeds

Parnidi1, I Santoso1, D Hariyanto1, M Sohri1, W M Mahayu1, T Hidayat RS1, M

Murianingrum1, M Machfud1, F Rochman1, Marjani1, G S A Fatah1, Supriyadi2,
and F Rahayu3
1. 1Research Center for Horticulture and Plantation, National Research Innovation Agency
2. 2Research Center for Behavioral and Circular Economics, National Research Innovation
Agency
3. 3Research Center for applied microbiology, National Research Innovation Agency

Email: parn003@brin.go.id@brin.go.id

Abstract. Agave seeds include growing seeds that have a low shelf life. Good storage will
maintain the quality and growth of Agave plant seeds. The purpose of this study is to get the
right agave plant seed storage method so that the quality and ability to grow are high. The
study was conducted in the greenhouse at the Indonesian Sweetener and Fiber Crops Research
Institute from January to September 2019. The research uses factorial-three factors based on a
completely randomized design (CRD) with 3 replications; each experimental unit consists of 5
plants. The first factor is the storage durations, consisting of 0, 21, and 35 days. The second
factor is the storage of seeds with cover and without a cover. The third factor is the reduction
in roots and leaves of the seeds, which consists of 1) normals root and leave, 2) cutting a half
root and normal leaves, 3) cutting a half root and leaves, 4) cutting the root and normal leaves,
and 5) cutting the roots and leaves. The results showed that the treatment of storage durations,
the use of cover, and the reduction of root organs and leaves affected agave plant seeds during
storage. After replanting, the seeds stored for up to five weeks do not show a significant
decrease in growth ability. The average ability to grow agave plant seeds after being stored for
three to five weeks is 100%. The distribution of agave seeds results from tissue cultures that
have been 3 months old at the development site can safely handle because the seeds can last up
to 35 days.
Keywords: Agave, Seeds, In vitro culture, Storage, Duration.

1. Introduction
Agave plants are known as one of the longest fiber-producing plants. Agave fiber is used to make
textiles, paper, rigging, handicrafts, and construction materials. According to Santoso (2009,
Parnidi and Setyo-Budi, 2016), the world's need for agave fiber reached 319,000 tons per year.
Agave fiber production from various countries in the world only reaches 281,800 tons per year, so
the need for agave fiber in the world cannot be fulfilled. The high global need for agave fiber
provides quite promising opportunities to develop agave plants.
Quality Agave plant seeds are needed to increase the productivity of agave plants. Propagation
of agave plant seeds can be done conventionally or non-conventionally. The conventional agave
plant's seeds are obtained from shoots and bulbils. One Agave plant that is more than 3 years old
has 5–15 shoots, depending on soil and climate conditions (Davis et al., 2021; Davis et al., 2019).
Bulbil is produced by agave plants that are over 5 years old. An agave plant can produce 1,000–
4,000 bulbil (Ochse et al., 1961), and after producing bulbil, the plant will die.
The propagation of non-conventional agave plant seeds on a large scale can be produced
through in vitro culture techniques with a fairly high success rate, close to 90% (Binh et al., 1990;
Parnidi and Setyo-Budi, 2016). Propagation of plant seeds with in vitro culture techniques is one
method for providing fast, healthy, pure, and uniform planting material (Parnidi and Setyo-Budi,
2016b; Ridhawati et al., 2017). In vitro culture techniques on agave plants produce small,
lightweight seeds that are easier to transport over long distances. An important factor that needs to
be considered in sending agave plant seeds is the delivery period from the seed production site to
the planting site. Agave plant seeds from in vitro culture require optimum conditions so the seeds
do not suffer damage. The distribution of agave plant seeds is a challenge in the development of
agave plants because the place of seed production is far from the garden. Efforts to increase
efficiency in shipping and maintain the life ability of agave plant seeds can be done directly or by
manipulating the environment of the seeds. This aims to maintain high seed viability during the
shipping process (Panuntun et al., 2017). The viability of agave plant seeds can be maintained by
reducing leaf and root tissue in the seeds. The deduction of root and leaf organs in seeds aims to
reduce the level of respiration and transpiration in agave plant seeds. If the place of seed
production is far from the garden, a good simulation of seed storage is needed so the seeds are not
damaged at the time of delivery. Based on the background above, it is necessary to research the
effect of storage treatments on the growth of in vitro agave (Agave sisalana L. perrine) seeds.

2. Materials and Methods

2.1 Ingredients and Research Time
Agave plant seeds from 10 in vitro culture experiments that were conducted for acclimatization up
to the age of three months were used as the study's materials. Sand media is used for planting on a
polybag that is 10 cm × 12.5 cm in size. From January to September 2019, the research was carried
out in the greenhouse of the Indonesian Sweeteners and Fiber Crops Research Institute.

2.2 Research Approach

The research uses factorial-three factors based on completely randomized design (CRD) with 3
replications, each experimental unit consists of 5 seeds. The first factor is the storage durations
consisting of 0, 21, and 35 days, the second factor is a cover and without a cover. The third factor
is the reduction of roots and leaves in the seeds, which consists of 1) normal root and leave, 2)
cutting a half root and normal leaves, 3) cutting a half root and leaves, 4) cutting the root and
normal leaves, and 5) cutting the roots and leaves.

2.3 Research Implementation

Before treatment, the fresh weight of agave plant seeds was measured to find out the initial fresh
weight of the seeds. Agave plant seeds are stored according to each treatment. The decrease in seed
weight, leaf length and width, root length, and the number of roots after storage were observed.
Furthermore, the seeds were replanted on a 5- by 10-inch pot tray for 45 HST. The media used
is compost, sand, and soil with a ratio of 3:1:1. Maintenance carries out according to the standard
cultivation of agave plants. After replanting, agave seed growth was observed to determine the
ability to recover agave plant seeds, including the fresh weight of the seeds, leaf length, root
length, and root amount. The data analysis uses the Analysis of Variation (ANOVA) at a 95%
confidence level.
 (a) (b) (c) (d) (e)
FIGURE 1. Conditions and shape of agave seeds before storing. (a) Normal agave seeds; (b) Agave
seeds with half roots and normal leaves; (c) Agave seeds with half roots and half leaves; (d) Agave
seeds without roots and normal leaves and (e) Agave seeds without roots and half leaves

3. Result and Discussion

3.1 General Conditions During Research
In general, the condition of agave plant seeds before being treated is good and healthy. The
average temperature and humidity at the time of research were 29.60% and 63.89%. The condition
of the seedlings after storage shows wrinkles, and some seeds have formed roots. Storage of agave
seeds for 5 weeks shows more wrinkled plants and roots that are formed longer than other
treatments.

3.2 The Effect of Treatment on The Growth of Agave Plant Seeds

Based on the results of the analysis, it appears that the growth of seedlings is not affected by the
interaction of closing treatment and the storage durations. The interaction of closure and storage
treatment affects the root length of agave plant seeds. The interaction of closing treatment with the
shape of the seeds affects the decrease in fresh weight and length of the agave plant seed roots but
does not affect on other growth variables (Table 1).

Table 1. Table of ANOVA interactions between closure treatment, storage duration and shape of
agave plant seeds propagated by in vitro culture.
Type III
Dependent Sum of Mean
Source Variable Squares df Square F Sig.
Corrected Model Plant height 469.96 29.00 16.21 11.57 0.00
Leave length 2.85 29.00 0.10 17.34 0.00
Total of leave 72.79 29.00 2.51 49.11 0.00
Root length 483.67 29.00 16.68 24.54 0.00
Total of root 1572.70 29.00 54.23 26.41 0.00
Fresh weight 20426.07 29.00 704.35 38.76 0.00
Intercept Plant height 0.38 1.00 0.38 0.27 0.61
Leave length 4.40 1.00 4.40 776.49 0.00
Total of leave 38.94 1.00 38.94 761.88 0.00
Root length 86.36 1.00 86.36 127.04 0.00
Total of root 144.91 1.00 144.91 70.56 0.00
Fresh weight 35753.88 1.00 35753.88 1968.00 0.00
Cover * Storage Plant height 152.39 2.00 76.19 54.38 0.00
 Leave length 0.19 2.00 0.10 16.84 0.00
Total of leave 0.41 2.00 0.21 4.03 0.02
Root length 6.90 2.00 3.45 5.07 0.01
Total of root 1.74 2.00 0.87 0.42 0.66
Fresh weight 256.71 2.00 128.36 7.06 0.00
Cover * Seeds Shap Plant height 19.73 4.00 4.93 3.52 0.01
Leave length 0.13 4.00 0.03 5.81 0.00
Total of leave 0.32 4.00 0.08 1.59 0.19
Root length 13.73 4.00 3.43 5.05 0.00
Total of root 4.60 4.00 1.15 0.56 0.69
Fresh weight 2.21 4.00 0.55 0.03 1.00
Storage * Seeds Shap Plant height 22.06 8.00 2.76 1.97 0.07
Leave length 0.14 8.00 0.02 3.18 0.01
Total of leave 18.26 8.00 2.28 44.65 0.00
Root length 132.94 8.00 16.62 24.45 0.00
Total of root 497.18 8.00 62.15 30.26 0.00
Fresh weight 78.35 8.00 9.79 0.54 0.82
Cover * Storage * Seeds Shap Plant height 23.38 8.00 2.92 2.09 0.05
Leave length 0.08 8.00 0.01 1.77 0.10
Total of leave 0.59 8.00 0.07 1.45 0.20
Root length 18.47 8.00 2.31 3.40 0.00
Total of root 24.64 8.00 3.08 1.50 0.18
Fresh weight 4.34 8.00 0.54 0.03 1.00
Cover Plant height 232.11 1.00 232.11 165.65 0.00
Leave length 0.02 1.00 0.02 3.31 0.07
Total of leave 0.18 1.00 0.18 3.48 0.07
Root length 10.60 1.00 10.60 15.59 0.00
Total of root 0.00 1.00 0.00 0.00 0.97
Fresh weight 490.64 1.00 490.64 27.00 0.00
Storage Plant height 0.31 2.00 0.16 0.11 0.90
Leave length 2.26 2.00 1.13 199.43 0.00
Total of leave 26.96 2.00 13.48 263.75 0.00
Root length 47.47 2.00 23.73 34.91 0.00
Total of root 72.65 2.00 36.32 17.69 0.00
Fresh weight 19444.53 2.00 9722.26 535.02 0.00
Seeds Shap Plant height 19.99 4.00 5.00 3.57 0.01
Leave length 0.02 4.00 0.01 1.05 0.39
Total of leave 26.07 4.00 6.52 127.51 0.00
Root length 253.58 4.00 63.40 93.26 0.00
Total of root 971.90 4.00 242.98 118.31 0.00
Fresh weight 149.30 4.00 37.32 2.05 0.10
Error Plant height 84.07 60.00 1.40
Leave length 0.34 60.00 0.01
 Total of leave 3.07 60.00 0.05
Root length 40.79 60.00 0.68
Total of root 123.23 60.00 2.05
Fresh weight 1090.32 60.00 18.17
Total Plant height 554.42 90.00
Leave length 7.59 90.00
Total of leave 114.80 90.00
Root length 610.82 90.00
Total of root 1840.84 90.00
Fresh weight 57270.27 90.00
Corrected Total Plant height 554.04 89.00
Leave length 3.19 89.00
Total of leave 75.86 89.00
Root length 524.46 89.00
Total of root 1695.93 89.00
Fresh weight 21516.40 89.00

The interaction of the storage durations and the shape of the seeds affects the number of leaves,
root length, and the number of roots of agave plant seeds. The interaction of closing treatment,
storage duration, and seed shape affects the length of agave plant seed roots (Table 1). The length
of the root is one important component for root growth, the root has a function to absorb nutrients,
water, and minerals (Theodore et al., 1997).
Agave plant seed fresh weight shrinkage is more influenced by agave plant seed closure and
shape. Agave plant seeds with fresh weight shrinkage after closure treatment are smaller than those
without closure. Closing treatment, combined with the treatment of reducing plant organs in the
roots and leaves, indicates an influence on the growth variables of agave plant seeds. The smallest
fresh weight shrinkage is found in the root treatment that is cut off, and the leaves are cut in half
with a cover with an average shrinkage of fresh weight of 33.28%. The largest fresh weight
shrinkage is found in the root treatment of half, and the leaves are not cut and are not given a
cover, with an average shrinkage of fresh weight of 39.65%.
The low shrinkage of fresh weight in the root treatment is cut off, and the leaves are cut in half
in a closed condition allegedly due to respiration that occurs at a smaller rate so that the fresh
weight shrinkage that occurs is small. Meanwhile, the root treatment is cut in half, and the normal
leaves in a condition that is not closed experience fresh weight shrinkage that is greater than other
treatments. The treatment without closure is unable to prevent water loss during storage. The use
of a cover can prevent agave plant seeds from losing wet weight. The weight loss of agave plant
seeds that are closed is lower than that without closure (Table 2 and Figure 2).
The agave seedlings were stored in conditions without a cover or with a cover, the growth of
agave seedlings propagated by tissue culture showed a decrease (Table 2). The decrease in agave
seedlings stored with cover was 18.62%, higher than without cover. The storage for 35 days
caused a decrease in agave seedlings propagated by tissue culture by 31.03%. Storage for 21 days
of agave seedling growth can reduce seedling growth by 23.04%. Agave seedlings from tissue
culture that were stored under normals root and leave conditions showed the greatest decrease in
growth (26.59%). The smallest decrease in seedling growth occurred when the seedlings were
stored in conditions of Cutting the root and normal leaves. In addition, under Cutting the root and
normal conditions, the roots grew longer and were followed by Cutting the roots and leaves
treatment.
 Agave seeds are included in the category of recalcitrant seeds. In general, recalcitrant seeds
cannot be stored below 10oC and above 35oC. Storage of recalcitrant seeds at low temperatures can
damage their viability (Panuntun et al., 2017). The respiration of agave seedlings increased due to
being placed in an open place so that they were less resistant to water loss due to transpiration,
compared to the treatment with a cover that only had a gap of less than 5% of the surface for
ventilation. Panuntun et al. (2017); Fauzan et al, (2017); Putra et al. (2021) stated that the
germination of single bud-set sugarcane seeds decreased after two days of storage without closure
at room temperature.
According to Santoso and Cholid (2019), Agave has a CAM (Crasulation Acid Metabolism)
type of carbon pathway that can grow well in dry conditions. The agave plant is classified as a
succulent, xeropitic, which allows the plant to survive in dry areas because of its small leaves,
thick cuticle, and hidden stomata. The stomata of agave plants close during the day to reduce the
evaporation of water, so the plants do not wither. The process of photosynthesis continues when
the sun shines brightly, with the help of CO2 taken at night.

Table 2. The effect of treatment on the growth of agave plant seeds

Variables Before After Decreases After Additional
Treatment storage storage seed after replanting of seeds
factors storage (%) growth
Storage with and without cover:
1. Plant height (cm) 12.95 11.41 11.89 18.44 61.66
2. Leave length (cm) 3.07 2.83 7.82 2.46 -12.96
Without 3. Total of leave 2.69 2.08 22.68 3.84 85.16
cover 4. Root length (cm) 2.86 2.23 22.03 11.91 434.61
5. Total of root 5.26 4.00 23.95 18.85 371.25
6. Fresh weight (g) 86.78 70.20 19.11 149.81 113.41
1.Plant height (cm) 13.10 14.78 -12.82 20.06 35.77
2. Leave length (cm) 3.03 2.81 7.26 2.49 -11.45
3. Total of leave 2.80 2.10 25.00 3.17 51.48
With cover
4. Root length (cm) 2.99 1.67 44.15 10.07 503.90
5. Total of root 5.41 4.13 23.66 19.07 361.55
6. Fresh weight (g) 92.16 69.61 24.47 153.96 121.17
Storage durations:
1.Plant height (cm) 10.79 10.79 0.00 17.98 66.62
2. Leave length (cm) 3.16 3.16 0.00 2.41 -23.65
3. Total of leave 2.74 2.74 0.00 3.56 30.17
0 day 2.90 2.90 10.46
4. Root length (cm) 0.00 260.84
5. Total of root 5.29 5.29 0.00 18.56 251.20
6. Fresh weight (g) 75.17 75.19 0.00 214.19 184.89
1.Plant height (cm) 14.32 14.37 0.00 19.82 37.93
2. Leave length (cm) 3.01 2.71 9.97 2.53 -6.63
3. Total of leave 2.53 1.89 25.30 3.43 81.34
21 days
4. Root length (cm) 3.01 1.80 40.20 12.00 565.68
5. Total of root 5.26 3.30 37.26 19.16 480.81
6. Fresh weight (g) 95.74 71.32 25.51 134.06 87.99
35 days 1.Plant height (cm) 13.96 14.12 -1.15 19.97 41.39
 2. Leave length (cm) 2.98 2.60 12.75 2.49 -4.11
3. Total of leave 2.97 1.63 45.12 3.53 117.21
4. Root length (cm) 2.88 1.14 60.42 10.50 822.67
5. Total of root 5.46 3.61 33.88 19.15 430.11
6. Fresh weight (g) 97.50 63.22 35.16 107.40 69.90
Reducing of roots and leaves:
1.Plant height (cm) 13.06 12.87 1.45 19.66 52.74
2. Leave length (cm) 3.06 2.83 7.52 2.44 -13.74
Normals
3. Total of leave 3.82 2.79 26.96 3.38 21.52
root and
leave 4. Root length (cm) 7.78 3.91 49.74 12.44 218.08
5. Total of root 9.91 4.99 49.65 20.03 301.67
6. Fresh weight (g) 98.08 74.29 24.26 160.48 116.03
1.Plant height (cm) 15.13 14.99 0.93 20.08 33.99
Cutting a 2. Leave length (cm) 3.19 2.99 6.27 2.56 -14.12
half root 3. Total of leave 3.73 2.60 30.29 3.44 32.46
and normal 4. Root length (cm) 3.26 2.19 32.82 11.28 414.72
leaves, 5. Total of root 8.34 5.34 35.97 19.05 256.56
6. Fresh weight (g) 99.17 77.35 22.00 168.31 117.61
1.Plant height (cm) 11.66 11.71 -0.43 18.17 55.17
2. Leave length (cm) 3.09 2.88 6.80 2.37 -17.73
Cutting a 1.23 1.23
3. Total of leave 0.00 3.83 210.78
half root
and leaves 4. Root length (cm) 3.59 2.29 36.21 11.11 385.67
5. Total of root 8.42 4.66 44.66 17.70 280.19
6. Fresh weight (g) 73.99 58.13 21.44 124.87 114.80
1.Plant height (cm) 14.18 13.80 2.68 19.82 43.59
Cutting the 2. Leave length (cm) 3.10 2.87 7.42 2.43 -15.29
root and 3. Total of leave 3.93 2.81 28.50 3.38 20.56
normal 4. Root length (cm) 0.00 0.97 -97.00 10.05 931.97
leaves 5. Total of root 0.00 3.09 -309.00 18.77 507.79
6. Fresh weight (g) 103.31 81.06 21.54 178.72 120.49
1.Plant height (cm) 11.10 12.10 -9.01 18.54 53.27
2. Leave length (cm) 2.81 2.53 9.96 2.57 1.53
Cutting the
3. Total of leave 1.00 1.00 0.00 3.50 250.00
roots and
leaves 4. Root length (cm) 0.00 0.37 -37.00 10.05 2617.84
5. Total of root 0.00 2.26 -226.00 19.24 753.18
6. Fresh weight (g) 72.79 58.71 19.34 127.04 116.39
Notes: a negative number (-) in Decreases seed after storage indicates that the seed has
increased growth.

Suhartono and Iskandar (2017) state that cabbage carried out storage in an open basket greater
decrease in fresh weight compared to storage using cardboard. O2 and CO2 balance also affect the
weight loss of agave plant seeds. Closing treatment reduces the rate of respiration, maintains sugar,
and reduces browning on the surface. The optimal level of O2 and CO2 is influenced by cultivars
and storage time (Almeida et al., 2018).
 A B C D E

1. The results of storage treatment, without cover at agave seeds

A B C D E
2. The results of storage treatment with cover at agave seeds
FIGURE 2. Agave seeds after being stored for 35 days
Treatment of closure, storage durations, and shape of agave plant seeds affect the length of the
root. In general, the closing treatment stored for 3 weeks shows better growth of root length than
other treatments. Meanwhile, agave plant seeds whose roots are cut off and the normal leaves
stored for 21 days and closed indicate the growth of seed roots that are the longest compared to
other treatments. Root growth shows faster growth compared to leaf formation. This is allegedly
because the roots develop first, so the process of root formation is faster than the formation of
shoots. Hartmann et al. (2002); Apriani and Suhartanto (2015); Hariani et al., (2020); Fransisica et
al., (20219); Fatima et al., (2019) state that the cuttings can first grow roots and then shoots, and
vice versa.

3.3 Recovery of Agave Plant Seeds After Transplanting

Based on the results of the analysis, it appears that the storage treatment affects the growth of
agave plant seeds after planting on soil media in a pot tray (transplanting). The treatment does not
affect the number of roots and wet weight of plants (Table 2).
After the seeds are planted for 45 days, the number of leaves, the number of roots, the length of
the roots, and the wet weight indicate that the variables are more influenced by the treatment. Plant
height and leaf width indicate they are not influenced by treatment. It is suspected that the storage
treatment has not reached the critical point for agave seeds. Hariani et al. (2022) state that
pineapple seedlings have decreased after 20 days of storage.
Agave seeds have good recovery power from the stress conditions experienced, which is shown
by the immediate return to normal growth. Storage for 35 days induces the best roots. The longer
the seeds are stored, after planting the number of roots formed becomes than other treatments. This
is allegedly related to the source of food acquisition by seeds. The treatment that results in a high
percentage of sprouts and deep roots will certainly increase food reserves, both from the process of
photosynthesis in the shoots and from the absorption of nutrients from the medium by the roots.

4. Conclusion
The treatment of closure, pruning of organs, and storage duration affect the decline in the growth of
agave plant seeds. Agave plant seeds have high recovery power. Agave plant seeds can survive being
stored for up to 35 days, either with or without cover. Reduction of root and leafe organs in agave
plant seeds can reduce the weight of the seed, which can reduce shipping costs.

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