Somatic embryo formation, plantlet vigorous and greenhouse preliminary test of garlic bulblet obtained through somatic embryogenesis

Garlic production in Indonesia is still low due to limited availability of seedling. Approximately 10% of garlic yield are used for the next cultivation. Therefore about 90 % of garlic consumption are still imported from China. In addition to conventional seed production, alternative routes are needed, such as through somatic embryogenesis. Research consists of three activities, namely 1) induction and multiplication of callus, 2) embryo somatic and plantlet formation and 3) preliminary test of bulblet in vitro in the greenhouse. Parameters observed in the laboratory activities were time of callus emergence, callus weight and morphology, time of somatic embryo emergence, embryo diameter and weight per bottle, plantlet number and vigorous and bulblet number per bottle, while in the greenhouse, parameter observed were survival of bulblet number, plant height, leaf number per plant, and clove number and weight. The result showed that application of 2,4-D 3 mg/l with casein hydrolysate acid was suitable for callus induction of garlic var. Lumbu Kuning, while BAP 2 mg/l was the most suitable formula for callus multiplication, the addition of paclobutrazol 1 ppm and sucrose 20% in MS medium was the most suitable treatment for plantlet vigorous, while the bulblet formation could be developed by using jasmonic acid with sucrose of 12%. The best medium for growjng media of in vitro bulblet in the greenhouse was the combination of sandy soil, cocopeat and manure.


Introduction
Garlic (Allium sativum) is an economic crop which has been consumed for seasoning in many foods and medicinal purposes.In Indonesia, garlic production is still low due to limited availability of seed.Approximately 10% of garlic yield are used for the next cultivation.Therefore about 90 % of garlic consumption are still imported from China.Other constraint, commercial garlic was sterile so they can only propagate vegetatively which affected a low of multiplication rate.Plant regeneration through conventional technique is very low, so the use of tissue culture especially somatic embryogenesis can be expected to produce seeds in large quantities.Somatic embryogenesis was a potential and prospective ways for garlic regeneration [1,2], but there was no standard protocol yet.The successful formation of somatic embryos is the key in multiplication through somatic embryogenesis.
Some protocols on embryogenesis somatic of garlic had been developed [3,4,5], but still needs improvement.Those various types of explants were used, it turns out that the use of root explants was the best [6].Addition of jasmonic acid 10 µm and sucrose 11 percent in MS containing NAA 0.5 ppm was the highest multiplication rate [7].Application of IAA 0.5 ppm and paclobutrazol produced healthy and survival

Callus induction, multiplication, and morphology
The time for callus emergence, callus number and weight are important parameters.The number of days for the first time of callus emergence was influenced by dosage of 2,4-D applied with casein hydrolysate acid.Table 1 showed that addition of 2,4-D applied with hydrolyzed acid (CH3) had a positive effect on callus formation from root explants.Among the five doses tested, the best in producing calluses was 2,4-D 3 mg/l + CH3 3mg/ l where the weight of calluses was the greatest (1.58 grams).Addition of kinetin to the 2,4-D treatment tend to slow down callus induction.Therefore, it is sufficient to use 2,4-D plus hydrolyze acid.Callus emergence time and its cumulative weight were influenced by dosage of 2,4-D added with casein hydrolysate acid (Table 1, Figure1).Different dosage of 2,4-D alone or in combination with Kinetin was significantly influencing the percentage of explants producing calli.All of the callus growth rate (GR) increases until dosage of 2,4-D 3 mg/l, with value of 1.58 g per bottle (Table 1), when kinetin was added, callus weight increase to 1.72 g per bottle at 10 weeks after planting (Table 2).

Somatic embryos formation and planlet vigorous
Data of the first emergence of green spot indicate that the use of BAP 3 mg/l was the fastest at 9 weeks after subculture (Figure 2a).It was a positive correlation with the highest number of somatic embryos with the average of 77.21 % (Figure 2b).The fastest time in somatic embryo formation also correlated with diameter of somatic embryo and plantlet number (Figure 2c and 2d).Addition NAA to 0.5% from 0.1% in media MS + BA 2 mg/l decreased somatic embryo diameter, and the number of plantlets per bottle (Figure 3a and 3b), although did not influence to improve plantlet vigorously (Figure 3c).For improving plantlet quality, the application of paclobutrazol in combination with NAA were applied.The result showed that application of NAA 0.5 combined with paclobutrazol 1 ppm did not increase the plantlet number (Figure 4a), but improved plantlets more vigorous (Figure 4b).This result is in line with previous research results [10,8].We hope that improving the vigorous of plantlet could be more easily acclimatized, but unfortunately the plantlet acclimatization was still not succeeded yet.Morphologically, the callus which was friable with a yellowish white colour has succeeded in forming shoots.Callus properties with the above characteristics can be obtained in Lumbu Kuning variety (Figure 1a and 1b).White callus with a crumb structure has the potential to become somatic embryos with marked formation of green spots from the callus (Figure 5a and 5b).After the callus is 4-5 months old, it proliferates so that with the appearance of green spots on some callus, both single and in colonies (Figure 5a and 5b).This callus showed that callus regeneration had occurred to become somatic embryos which then develops into shoots.At the age of one month later these shoots continued to develop into new planlet.Some of these shoots already had roots, but some must be subcultured first in rooting media (Figure 5c and 5d).

Bulbet formation and acclimatization
One of the constrain in the garlic micropropagation program was the success rate of acclimatization of plantlets produced through somatic embryogenesis techniques.Very thin leaf and weak stem structures of planlet which under ideal conditions in the laboratorium will easily become brittle when planted in open natural conditions.To overcome this, it was necessary to improve the level of plantlet vigorous.Figure 4b indicate that the use of paclobutrazole could improve planlet vigorous.Application NAA 0.5 with paclobutrazol 0.1 ppm produces more vigorous plants rather than NAA with 0.1 paclobutrazol 0.5 ppm.It is in line with previous research of result [10].An effort to increase the success of planlet acclimatization is to make bulbets in vitro, due to availability of energy source for planlet during in acclimatization (Figure 6a and 6b).
For acclimatization, plantlet and bulblet need the porous soil media to produce the best bulb, therefore the type of sandy soil was more suitable to obtain good growth.Five types of media (soil, sand, manure and cocopeat) were tested in singgle or in combination treaments.The result showed that composition of complete media consist of a mixture of soil, sand, manure and cocopeat showed best growth especially in percentage of bulbet growth, plant height, and leaf number (Table 3).Treatment of jasmonic acid and 11 % sucrose increased the formation of bulblets (Figure 6a and 6b), and these bubblets were successfully acclimatized in the greenhouse (Figure 6c).

Conclussion
Application of 2,4-D 3 mg/l with casein hydrolysate acid was the best formula for callus induction of garlic var.Lumbu Kuning, while BAP 2 mg/l was suitable formula for callus multiplication, the addition of paclobutrazol 1 ppm and sucrose 20% in MS medium was the best treatment for plantlet vigorous, while The bulblet formation could be developed by using jasmonic acid with sucrose of 12%.The suitable medium

Figure 2 .Figure 3 .Figure 4 .Figure 5 .
The effect of BAP on somatic embryos development and plantlet number: (a) time of green spot emergence, (b) percentage of embryo number, (c) diameter of embryo per bottle, and (d) plantlet number per bottle.Effect of BA and NAA to embryo somatic and plantlet development: (a) somatic embryo diameter, (b) plantlet number per bottle; (c) plantlet vigorous.a b Effect of NAA and Paclobutrazol on plantlet: (a) plantlet number per bottle, (b) plantlet vigorous.Proliferation and elongation stage of somatic embryo of garlic: (a) somatic embryos with green dots , (b) somatic embryos with bigger green dot, (c) shoot elongation, (d) planlets.

Table 2 .
Effect of 2,4-D and kinetin on callus induction time from root explants, callus weight after 10 weeks, and callus morphology of Lumbu Kuning variety on MS media added with CH3 3 mg/l.

Table 3 .
In vitro bulblet acclimatization test of garlic on various growth media compositions.