Hormone-injected leaf cutting, a new efficient in vivo multiplication protocol for two succulent plants

Xiaodan Xu 1 and Wei Zheng 2, *

1 Faculty of Art and Communication

Kunming University of Science and Technology

Kunming 650500

China

2 Faculty of Architecture and City Planning

Kunming University of Science and Technology

Kunming 650500

China

* Corresponding author:

E-mail: [email protected].

1 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 Abstract: This study aimed to establish a simple and efficient in vivo

multiplication protocol by leaf cutting to satisfy the supply of young succulent

ornamentals Pachyveria pachytoides and Sedum morganianum. The regenerability of

leaves injected with 6-benzylaminopurine (BAP) and α-naphthalene acetic acid (NAA)

in vivo were tested with common leaf cutting as control. Results showed a 100%

shoot induction frequency using hormone-injeceted methods for the two species. The

number of shoots per leaf of 4.0 or 6.0 mg l−1 BAP and 0.1 mg l−1 NAA injected in

vivo (5.08-5.14 in P. pachytoides, and 6.22-6.74 for S. morganianum) were

significantly greater than that of the other treatments. Since the hormone-injected leaf

cutting needs no aseptic operation which is necessary for in vitro multiplication, it is

simple for the commercial production of the two species. The new in vivo propagation

method would be of great interest for growers and breeders of succulent plants.

Keywords: Pachyveria pachytoides, Sedum morganianum, bud multiplication, direct

organogenesis

2 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 1. Introduction

In vitro proliferation is currently applied to propagate many ornamental plants

(Jaykuma et al., 2013; Ozel et al., 2008; Chikkala et al., 2009), but commercial

production requires the use of cost-effective in vivo multiplication.

Succulent plants are characterized by thick and fleshy parts to retain water

in arid climates or soil conditions (Nyffeler and Eggi, 2010). They are grown

as ornamental plants because of their striking and unusual appearance. The family

Crassulaceae is a large group of succulent plants (Griffiths, 2013) with several genera,

such as Echeveria (Borys and Leszczynska-Borys, 2013), Sedum (Sendo et al., 2007),

Kalanchoe (Heiko et al., 2014), and Pachyveria; in this family of plants, species and

progenies vary in terms of the size of their rosette leaves, color, fatty leaves, and

length of vegetative and reproductive organs, as well as ease of cultivation (Rafael et

al., 2013).

Leaf cutting is one of the main propagation methods for succulent plants. One

(seldom two) bud and multiple roots can be directly germinated from petioles via leaf

cutting. Compared with that of hormone-supplemented in vitro propagation, the

multiplication time of common leaf cutting is very low.

Here, we speculate that better in vivo multiplication via bud formation can be

achieved if the fatty leaves were considered as the basic MS medium (Murashige and

Skoog, 1962) and the hormones were injected into the mesophyll cells of these leaves.

However, no detailed reports have been presented regarding hormone treatment in

succulent plants in vivo.

3 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 We aimed to establish a simple and efficient in vivo proliferation protocol for

succulent plants by using leaf cutting of Pachyveria pachytoides and Sedum

morganianum, which are members of the family Crassulaceae endemic to Mexico and

cultivated worldwide as ornamental plants (José et al., 2010), to verify our hypothesis.

The research would be useful for commercial production and further research of

succulent plants.

2. Materials and Methods

2.1 Materials

Plants of P. pachytoides and S. morganianum were bought from Dounan Flower

Market, Kunming, Yunnan Province, China and cultivated in a greenhouse in

Kunming University of Science and Technology.

2.2 Direct organogenesis from hormone-injected in vivo leaves

The mature leaves of the two species were excised from their mother plants and

dried in the shade for 3 d. The average leaf volume (3.0 ml for P. pachytoides and 0.6

ml for S. morganianum) and its solution-absorbing capacity (0.10 ml for P.

pachytoides and 0.02 ml for S. morganianum) were measured. Afterward, the

hormone concentrations were calculated (60.0, 120.0, and 180.0 mg l−1 BAP; 3.0

mg l−1 NAA) to obtain the final hormone concentration of 2.0, 4.0, and 6.0 mg l−1

BAP and 0.1 mg l−1 NAA in the leaves after injection. Injection was made near the

petiole and deep into the mid-blade. With the adaxial side facing upward, the injected

4 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 leaves were then planted in a mixture of peat, vermiculite, and perlite (1:1:1) in a Petri

dish.

2.3 Experimental design and statistical analyses

All of the experiments were conducted in three repetitions (20 explants per

repetition) by a randomized complete block design. Common leaf cutting without

hormone treatment was performed as control. All of the cultures were maintained in a

growth chamber at 20 °C to 25 °C (Nishida et al., 2009) under cool, white fluorescent

lamps (45 μmol s−1 m−2) with 14 h photoperiod. The number of days to shoot or root

initiation for each treatment was recorded. The average number of adventitious shoots

or roots induced per leaf and the induction frequency were measured after 30 d of

culture.

Data in the form of percentages were subjected to arcsine transformation

whereas non-transformed data are presented in tables. Data were analyzed by the

analysis of variance (ANOVA) and Duncan’s multiple range test with a 0.05

confidence interval.

3. Results

Adventitious shoots and roots originated directly from parenchyma cells near the

vascular bundle sheath of in vivo and in vitro propagated leaves of P. pachytoides and

S. morganianum. The buds of P. pachytoides and S. morganianum were initiated

5 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 around 7 and 15 d after propagation, respectively. In addition, these two succulent

leaves developed roots around 18 d after propagated.

In the culture of two succulent plants, the shoot induction frequency was 100%

for all the treatments (Table 1). The number of shoots per leaf (5.08-5.14 in P.

pachytoides, and 6.22-6.74 for S. morganianum) of 4.0 or 6.0 mg l−1 BAP and 0.1 mg

l−1 NAA injected in vivo was significantly greater than that of the other treatments

(Figure 1). While, the root induction frequency by hormone-injected methods was

lower than that of common cutting (100%). In addition, the number of roots decreased

with the increasing of BAP concentration. This result indicated that a higher

concentration of BAP could induce bud formation than root formation.

4. Discussion

These succulent ornamental plants can be sold in the market as small plants (1

cm to 3 cm in height), even without roots. Therefore, this study mainly aimed to

promote multiple bud induction and proliferation rather than root formation. In this

experiment, 4.0:0.1 BAP:NAA hormone injected leaf cutting could be used for

multiplication of P. pachytoides and S. morganianum because the induction of shoots

was the highest in this treatment, as well as the merits of cheap and simple.

Compared with in vitro propagation, hormone-injected in vivo propagation did

not require aseptic operation, thereby reducing workload and production cost.

Therefore, hormone-injected in vivo leaf cutting is an effective method for the

6 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 commercial production of P. pachytoides and S. morganianum, even other succulent

plants.

Further studies could be conducted to exploit the potential of mutation and

genetic transformation for the improvement of succulent ornamentals by using this

new hormone-injected in vivo propagation.

References

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Acknowledgments

This work was supported by the National Natural Science Foundation of China

(Grant No. 31360488).

8 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 Figure legends

Fig.1. Direct organogenesis from the leaves of Pachyveria pachytoides (A, B) and

Sedum morganianum (C, D) after 30 days of propagation. (A, C) Common leaf

cutting; (B, D) hormone (4.0 mg l−1 BAP + 0.1 mg l−1 NAA)-injected in vivo leaves.

Fig. 1.

9 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017 1 Table 1 Effect of different method on direct organogenesis of Pachyveria pachytoides

2 and Sedum morganianum.

Species BAP + Shoot No. of Root No. of

NAA induction shoots/leaf induction roots/leaf

(mg l-1) (%) (%)

Pachyveria pachytoides 0 + 0 100.00 a 1.16 ± 0.12 c 100.00 a 8.13 ± 0.92 a

2 + 0.1 100.00 a 3.79 ± 0.40 b 92.67 a 1.05 ± 0.13 b

4 + 0.1 100.00 a 5.14 ± 0.36 a 33.65 ab 0.44 ± 0.03 c

6 + 0.1 100.00 a 5.08 ± 0.41 a 10.24 b 0.12 ± 0.01 c

Sedum morganianum 0 + 0 100.00 a 1.37 ± 0.15 c 100.00 a 5.62 ± 0.78 a

2 + 0.1 100.00 a 3.93 ± 0.41 b 90.00 a 2.51 ± 0.32 b

4 + 0.1 100.00 a 6.74 ± 0.58 a 83.67 a 1.90 ± 0.20 c

6 + 0.1 100.00 a 6.22 ± 0.61 a 82.10 a 1.82 ± 0.21 c

3 Values within a column for each species followed by the same letter are not

4 significantly different at p=0.05 by Duncan’s multiple range test.

5

10 PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2656v2 | CC BY 4.0 Open Access | rec: 7 Mar 2017, publ: 7 Mar 2017